Team:Imperial College/mainresults

From 2013.igem.org

(Difference between revisions)
Line 17: Line 17:
<div id="CollapsiblePanelM11" class="CollapsiblePanel">
<div id="CollapsiblePanelM11" class="CollapsiblePanel">
-
<div class="CollapsiblePanelTab" tabindex="0"><h4><i>E. coli</i> breaks down PUR     </html><font size="1">&#9660;</font size="1"><html></h4></div>
+
<div class="CollapsiblePanelTab" tabindex="0"><h4>We made P3HB bioplastic     </html><font size="1">&#9660;</font size="1"><html></h4></div>
<div class="CollapsiblePanelContent">
<div class="CollapsiblePanelContent">
</html>
</html>
-
<p>We proved the PUR esterase activity of EstCS2 in colourimetric assay with the substrate analog para-Nitrophenyl butyrate. This result is important because we now have enzyme to break down a common material in mixed plastic waste.</p>
+
<p>We used <i>E. coli</i> that expresses PhaCAB enzymes  to make P3HB bioplastic. This is tested on nile red plates which stains P3HB. </p>
-
[[File: 4-NP_abs02.png|thumbnail|center|800px|The increase in absorbance that accompanies the cleavage of para-Nitrophenyl butyrate by PUR Esterase EstCS2. Figure by Imperial College London iGEM 2013.]]
+
{| class="wikitable" style="margin: 1em auto 1em auto;"
-
[[File: PUREstC2_graph.png|thumbnail|center|700px|The concentration of 4-Nitrophenol released by PUR Esterase EstCS2 activity. Empty Vector and Substrate alone were used as negative controls. Figure by Imperial College London iGEM 2013.]]
+
|[[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.</b>]]
 +
|}
Line 30: Line 31:
</div>
</div>
</div>
</div>
 +
<div id="CollapsiblePanelM12" class="CollapsiblePanel">
<div id="CollapsiblePanelM12" class="CollapsiblePanel">
-
<div class="CollapsiblePanelTab" tabindex="0"><h4><i>E. coli</i> grows on ethylene glycol      </html><font size="1">&#9660;</font size="1"><html></h4></div>
 
-
<div class="CollapsiblePanelContent">
 
-
 
-
</html>
 
-
<p align="justify>We tested cell growth in ethylene glycol, which is a PUR degradation product. We showed that at 37°C, cell growth is not significantly affected by the concentration of ethylene glycol. Thus our engineered bacteria can grow in the bioreactors!</p>
 
-
[[File: EG_growth.png|thumbnail|center|800px|Ethylene glycol in LB with Stress Response cells. <i>E. coli</i>MG1655 were grown in ethylene glycol, a byproduct of polyurethane degradation. Cells were grown in 0mM, 100mM or 200mM Ethylene Glycol. At 37°C, the concentrations of ethylene glycol used do not affect growth, however at 30°C, the increasing concentration results in halved growth. A two-tailed t-test addressing the null hypothesis, temperature does not affect growth with ethylene glycol shows that the null hypothesis must be rejected as p = 0.001. Data points show final time point after 6h growth for each concentration. Growth was at 37°C and 30°C with shaking over 6h. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.]]
 
-
 
-
 
-
<html>
 
-
</div>
 
-
</div>
 
-
 
-
<div id="CollapsiblePanelM13" class="CollapsiblePanel">
 
<div class="CollapsiblePanelTab" tabindex="0"><h4>Modelling      </html><font size="1">&#9660;</font size="1"><html></h4></div>
<div class="CollapsiblePanelTab" tabindex="0"><h4>Modelling      </html><font size="1">&#9660;</font size="1"><html></h4></div>
<div class="CollapsiblePanelContent">
<div class="CollapsiblePanelContent">
Line 56: Line 45:
</div>
</div>
-
<div id="CollapsiblePanelM14" class="CollapsiblePanel">
+
<div id="CollapsiblePanelM13" class="CollapsiblePanel">
-
<div class="CollapsiblePanelTab" tabindex="0"><h4>Optimised bioplastic production      </html><font size="1">&#9660;</font size="1"><html></h4></div>
+
<div class="CollapsiblePanelTab" tabindex="0"><h4>We optimised bioplastic production      </html><font size="1">&#9660;</font size="1"><html></h4></div>
<div class="CollapsiblePanelContent">
<div class="CollapsiblePanelContent">
Line 76: Line 65:
</div>
</div>
-
<div id="CollapsiblePanelM15" class="CollapsiblePanel">
+
<div id="CollapsiblePanelM14" class="CollapsiblePanel">
-
<div class="CollapsiblePanelTab" tabindex="0"><h4>Bioplastic from mixed waste      </html><font size="1">&#9660;</font size="1"><html></h4></div>
+
<div class="CollapsiblePanelTab" tabindex="0"><h4>We made bioplastic from mixed waste      </html><font size="1">&#9660;</font size="1"><html></h4></div>
<div class="CollapsiblePanelContent">
<div class="CollapsiblePanelContent">
Line 148: Line 137:
var CollapsiblePanelM11 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM11", {contentIsOpen:false});
var CollapsiblePanelM11 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM11", {contentIsOpen:false});
var CollapsiblePanelM12 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM12", {contentIsOpen:false});
var CollapsiblePanelM12 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM12", {contentIsOpen:false});
-
var CollapsiblePanelM13 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM13", {contentIsOpen:false});
+
var CollapsiblePanelM13 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM13", {contentIsOpen:True});
-
var CollapsiblePanelM14 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM14", {contentIsOpen:false});
+
var CollapsiblePanelM14 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM14", {contentIsOpen:True});
-
var CollapsiblePanelM15 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM15", {contentIsOpen:false});
+
 
var CollapsiblePanelM21 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM21", {contentIsOpen:false});
var CollapsiblePanelM21 = new Spry.Widget.CollapsiblePanel("CollapsiblePanelM21", {contentIsOpen:false});

Revision as of 16:53, 4 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. 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 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).

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 grows in P3HB and 3HB

Content

E. coli feeds on 3HB

Content

E. coli degrades P3HB

Content

Modelling P3HB degradation

Content

PLA degraded by E. coli

Content

Our Sponsors

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