Team:Imperial College/mainresults
From 2013.igem.org
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- | |[[File:Results-nile_red_plates.PNG|thumbnail|left|900px| <b> <i>E.coli</i> 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. | + | |[[File:Results-nile_red_plates.PNG|thumbnail|left|900px| <b> <i>E.coli</i> 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. </b>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.]] |
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- | <p align="justify">One of the objectives of [https://2013.igem.org/Team:Imperial_College/Waste_Degradation:_SRF Module 1] is to produce P3HB bioplastic from waste. We compared P3HB degradation by measuring the concentration of 3HB monomers, using P3HB we bought from sigma, we produced from glucose, and we produced from the waste collected from Powerday. We found that there is no significant difference among them. Thus <b>we conclude that we have made P3HB bioplastic from waste</b>.</p> | + | <p align="justify">One of the objectives of [https://2013.igem.org/Team:Imperial_College/Waste_Degradation:_SRF Module 1] is to produce P3HB bioplastic from waste. We compared P3HB degradation by measuring the concentration of 3HB monomers, using P3HB we bought from sigma, we produced from glucose, and we produced from the waste collected from Powerday. We found that there is no significant difference among them (p=0.1). Thus <b>we conclude that we have made P3HB bioplastic from waste</b>.</p> |
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<p align="justify">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. <b>We are the first iGEM team to degrade bioplastics!</b></p> | <p align="justify">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. <b>We are the first iGEM team to degrade bioplastics!</b></p> | ||
- | [[File:Imperial_clearing_zone.jpg|thumbnail|centre|950px|<font size="2.7">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.]] | + | [[File:Imperial_clearing_zone.jpg|thumbnail|centre|950px|<font size="2.7"><b>PhaZ1 showing P3HB degradation ability by generating a clear zone on a P3HB LB agar plate.</b> 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.]] |
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- | <div class="CollapsiblePanelTab" tabindex="0"><h4>We | + | <div class="CollapsiblePanelTab" tabindex="0"><h4>We can degrade PLA </html><font size="1">▼</font size="1"><html></h4></div> |
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+ | We used scanning electron microscopy, a common method in plastics studies, to visualise changes on PLA bioplastic surface. Proteinase K we bought from Sigma Aldrich has shown promising result in creating pores on PLA surface. PLA surface treated with lysed cells expressing proteinase K has also shown slight some differences after 3 days. | ||
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- | |[[File:PLA_SEM_all_ICL.jpg|thumbnail|left|600px|Scanning Electron Micrographs show the increased pitting of PLA surface after enzyme and cell lysate treatment. | + | |[[File:PLA_SEM_all_ICL.jpg|thumbnail|left|600px|<b>Scanning Electron Micrographs show the increased pitting of PLA surface after enzyme and cell lysate treatment.</b> Images by Imperial College iGEM Team 3013. |
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Revision as of 00:36, 5 October 2013
Main Results
Resource-full Waste
Plastic Fantastic
We made P3HB bioplastic ▼
We used E. coli that expresses PhaCAB enzymes to make P3HB bioplastic. P3HB produced in cells were stained with Nile Red for fluorescent microscope imaging, and strong fluorescence was detected at the area where PhaCAB-expressing cells were streaked.
Our model predicts increased PhaB expression boosts P3HB production ▼
In order to realistically improve our experimental design, we produced P3HB synthesis model with considerations of central metabolic pathway within the cells. Our model predicts that during the synthesis of P3HB, the concentration of PhaB enzymes is an important rate limiting factor that can actually be regulated.
We optimised P3HB bioplastic production ▼
Since our model predicts that P3HB production can be significantly improved by expressing more P3HB polymerase PhaB, we designed a [http://parts.igem.org/Part:BBa_K1149051 hybrid promoter] which, 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.
We made bioplastic from mixed waste ▼
One of the objectives of Module 1 is to produce P3HB bioplastic from waste. We compared P3HB degradation by measuring the concentration of 3HB monomers, using P3HB we bought from sigma, we produced from glucose, and we produced from the waste collected from Powerday. We found that there is no significant difference among them (p=0.1). Thus we conclude that we have made P3HB bioplastic from waste.
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!
![](/wiki/images/6/68/Imperial_clearing_zone.jpg)
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 can degrade PLA ▼