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Team:SDU-Denmark/Tour50
From 2013.igem.org
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<h2>Results </h2> | <h2>Results </h2> | ||
| - | <h4> | + | <h4>If it looks like rubber and smells like rubber, it probably is rubber</h4> |
<p> | <p> | ||
| - | <span class="intro"> | + | <span class="intro">The following pages</span> thoroughly present our data. The hours of work in the lab were easily justified by the excitement of new results, and we hope that you immerse yourself. We invite you to dig deeper to view our results and to experience our excitement. But before you do that, this page points to our main accomplishments and presents a quick glance at the most cherished of our results: the production of Bacteriorganic Rubber. |
| - | + | ||
</p><p> | </p><p> | ||
| + | To quickly recap, we had two subprojects: optimization of the MEP pathway to increase the amount of building blocks (<span class="tooltipLink">IPP</span> <span class="tooltip"><span class="tooltipHeader">IPP</span>Isopentenyl pyrophosphate</span> and <span class="tooltipLink">DMAPP</span><span class="tooltip"><span class="tooltipHeader">DMAPP</span>Dimethylallyl pyrophosphate</span>) for rubber synthesis; and the introduction of the rubber polymerization enzyme HRT2 prenyltransferase of <span class="specialWord">Hevea brasiliensis</span>, into the <span class="specialWord">E. coli</span> bacteria. First, let's look at the latter. | ||
| - | + | </p> | |
| - | + | ||
| - | + | <p> | |
| + | <a class="popupImg alignRight" style="width:200px" href="https://static.igem.org/mediawiki/2013/b/bb/SDU2013_Rubber_Production_1.png" title=" Figure 1 - H<sup>1</sup>-NMR peak that suggests rubber production. Consult our characterization site for more info."> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/b/bb/SDU2013_Rubber_Production_1.png" style="width:200px" /> | ||
| + | Figure 1. | ||
| + | </a> | ||
| + | <span class="intro">Rubber synthesis</span><br> | ||
| + | The HRT2 prenyltransferase of <span class="specialWord">Hevea brasiliensis</span> was successfully introduced into the <span class="specialWord">E. coli</span> bacteria. H<sup>1</sup>-NMR was performed to detect rubber produced by our bacteria in comparison with a commercially purchased polyisoprene standard as the positive control and wild type cells <span class="specialWord">E. coli</span> as the negative control. The results show strong indication that we have indeed made rubber-producing bacteria <b>(Fig. 1)</b>. A second round of H<sup>1</sup>-NMR stated that the result was reproducible, and therefore we proudly announce that our CPS bacteria produce rubber; Bacteriorganic Rubber! | ||
| - | <span class=" | + | <span class="sourceReference">Bacteriorganic Rubber!</span> |
| - | + | <span class="tooltip"> | |
| + | <span class="tooltipHeader">Source:</span> | ||
| + | |||
| + | Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183 | ||
| - | </ | + | <a href="http://edu.cengage.co.uk/catalogue/product.aspx?isbn=0538734183" target="_blank">(Link)</a> |
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| - | |||
| - | |||
| - | + | </span> | |
| - | + | ||
</p><p> | </p><p> | ||
| + | <span class="intro">Optimization of MEP pathway</span><br> | ||
| + | We attempted to optimize the MEP pathway by overexpressing the genes (<span class="tooltipLink">Dxs</span><span class="tooltip"><span class="tooltipHeader">DXS</span>1-deoxyxylulose-5-phosphate synthase</span> and <span class="tooltipLink">IspG</span> <span class="tooltip"><span class="tooltipHeader">IspG</span>1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase</span>) which our model predicted to be rate limiting. Unfortunately, we were unsuccessful in building a construct containing <span class="specialWord">ispG</span>, and therefore we could not test the consequences of overexpressing this particular gene. However, we did successfully build a construct that overexpressed Dxs. In order to verify whether IPP and DMAPP concentrations increased by overexpression of the <span class="specialWord">dxs</span> gene, we performed gas chromatography (GC), though we were unable to prove or disprove the hypothesis. | ||
| + | <span class="sourceReference">the hypothesis.</span> | ||
| + | <span class="tooltip"> | ||
| + | <span class="tooltipHeader">Source:</span> | ||
| + | |||
| + | Fisher AJ, Rosenstiel TN, Shirk MC, Fall R. Nonradioactive assay for cellular dimethylallyl diphosphate. Anal Biochem. 2001 May 15;292(2):272-9. | ||
| - | < | + | <a href="http://www.ncbi.nlm.nih.gov/pubmed/11355861" target="_blank">(Link)</a> |
| - | + | ||
| - | </ | + | </span> |
| - | |||
| - | <a class="popupImg alignRight" style="width: | + | </p> |
| - | <img src="https://static.igem.org/mediawiki/2013/ | + | |
| + | <p> | ||
| + | |||
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| + | |||
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| + | <span class="intro">Controllable constructs</span><br> | ||
| + | |||
| + | <a class="popupImg alignRight" style="width:150px" href="https://static.igem.org/mediawiki/2013/3/37/SDU2013_Results_Controlable_1.png" title="Figure 2 - <i>HRT2</i> mRNA levels and Dxs protein levels were measured before and after addition of induction molecules (arabinose and IPTG, respectively). The results clearly prove that we can induce high expression of the genes upon addition of the induction molecules. View our characterization for much more details and results on the experiments."> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/3/37/SDU2013_Results_Controlable_1.png" style="width:150px" /> | ||
Figure 2. | Figure 2. | ||
</a> | </a> | ||
| - | + | An important part of the design of our constructs was the capability to control the expression of genes converting the fast growing bacteria to rubber producing bacteria. We therefore placed the expression of Dxs under the control of the lac-promoter and the expression of HRT2 under the control of the ara-promoter. We successfully proved that addition of IPTG induces the expression of Dxs, and addition of arabinose induces the expression of HRT2 <b>(Fig. 2)</b>. A cell viability experiment showed that the rubber producing bacteria were not growth impaired or in other ways toxically affected as otherwise expected. This surprising result might be due to the low amounts of rubber produced in the cells. Nevertheless, rubber production is taking place! | |
| + | </p><p> | ||
| + | <span class="intro">Added BioBrick parts and devices</span><br> | ||
| + | One of the ideals of iGEM is the vision of a large registry, where all imaginable DNA sequences usable for a synthetic microbiologist can be found in easy-to-use standardized parts. During our project, we have added 4 new basic parts and 13 devices to this registry. Dig deeper to explore the different bricks and submitted parts. | ||
</p><p> | </p><p> | ||
| + | <span class="intro">A different wiki</span><br> | ||
| + | One of our stated goals during this year’s iGEM competition was to invent a novel and intuitive approach to the iGEM wiki. We hope to reach a wide and varied audience and our wiki is part of that effort. Through a lot of hard work and great feedback, we are confident that our goal has been accomplished and that our tour is to your liking, dear reader. | ||
| - | |||
| - | </p | + | </p><p> |
| - | <span class="intro"> | + | <span class="intro">Jamboree result</span><br> |
| + | At the Regional Jamboree in Lyon our project achieved a Gold medal, advancement to the World Championship in Boston, and even a special award for Most Improved Registry Part. | ||
</p><p> | </p><p> | ||
| - | <span class="intro"> | + | Alongside all these achievements, we learned more about the hard work it requires to work together in large groups and received invaluable lab and life experiences. |
| + | |||
| + | </p><p> | ||
| + | |||
| + | <span class="intro">Dig deeper</span> to see all our results in details. Or go to the <span class="intro">next chapter</span> to see the future perspectives of our project. | ||
</p> | </p> | ||
| + | |||
Latest revision as of 02:05, 29 October 2013
Results
If it looks like rubber and smells like rubber, it probably is rubber
The following pages thoroughly present our data. The hours of work in the lab were easily justified by the excitement of new results, and we hope that you immerse yourself. We invite you to dig deeper to view our results and to experience our excitement. But before you do that, this page points to our main accomplishments and presents a quick glance at the most cherished of our results: the production of Bacteriorganic Rubber.
To quickly recap, we had two subprojects: optimization of the MEP pathway to increase the amount of building blocks (IPP IPPIsopentenyl pyrophosphate and DMAPPDMAPPDimethylallyl pyrophosphate) for rubber synthesis; and the introduction of the rubber polymerization enzyme HRT2 prenyltransferase of Hevea brasiliensis, into the E. coli bacteria. First, let's look at the latter.
Figure 1.
Rubber synthesis
The HRT2 prenyltransferase of Hevea brasiliensis was successfully introduced into the E. coli bacteria. H1-NMR was performed to detect rubber produced by our bacteria in comparison with a commercially purchased polyisoprene standard as the positive control and wild type cells E. coli as the negative control. The results show strong indication that we have indeed made rubber-producing bacteria (Fig. 1). A second round of H1-NMR stated that the result was reproducible, and therefore we proudly announce that our CPS bacteria produce rubber; Bacteriorganic Rubber!
Bacteriorganic Rubber!
Source:
Donald L. Pavia and Gary M. Lampman. Introduction to Spectroscopy, International Edition 4e (Book) ISBN-13: 9780538734189 / ISBN-10: 0538734183
(Link)
Optimization of MEP pathway
We attempted to optimize the MEP pathway by overexpressing the genes (DxsDXS1-deoxyxylulose-5-phosphate synthase and IspG IspG1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase) which our model predicted to be rate limiting. Unfortunately, we were unsuccessful in building a construct containing ispG, and therefore we could not test the consequences of overexpressing this particular gene. However, we did successfully build a construct that overexpressed Dxs. In order to verify whether IPP and DMAPP concentrations increased by overexpression of the dxs gene, we performed gas chromatography (GC), though we were unable to prove or disprove the hypothesis.
the hypothesis.
Source:
Fisher AJ, Rosenstiel TN, Shirk MC, Fall R. Nonradioactive assay for cellular dimethylallyl diphosphate. Anal Biochem. 2001 May 15;292(2):272-9.
(Link)
Controllable constructs
Figure 2.
An important part of the design of our constructs was the capability to control the expression of genes converting the fast growing bacteria to rubber producing bacteria. We therefore placed the expression of Dxs under the control of the lac-promoter and the expression of HRT2 under the control of the ara-promoter. We successfully proved that addition of IPTG induces the expression of Dxs, and addition of arabinose induces the expression of HRT2 (Fig. 2). A cell viability experiment showed that the rubber producing bacteria were not growth impaired or in other ways toxically affected as otherwise expected. This surprising result might be due to the low amounts of rubber produced in the cells. Nevertheless, rubber production is taking place!
Added BioBrick parts and devices
One of the ideals of iGEM is the vision of a large registry, where all imaginable DNA sequences usable for a synthetic microbiologist can be found in easy-to-use standardized parts. During our project, we have added 4 new basic parts and 13 devices to this registry. Dig deeper to explore the different bricks and submitted parts.
A different wiki
One of our stated goals during this year’s iGEM competition was to invent a novel and intuitive approach to the iGEM wiki. We hope to reach a wide and varied audience and our wiki is part of that effort. Through a lot of hard work and great feedback, we are confident that our goal has been accomplished and that our tour is to your liking, dear reader.
Jamboree result
At the Regional Jamboree in Lyon our project achieved a Gold medal, advancement to the World Championship in Boston, and even a special award for Most Improved Registry Part.
Alongside all these achievements, we learned more about the hard work it requires to work together in large groups and received invaluable lab and life experiences.
Dig deeper to see all our results in details. Or go to the next chapter to see the future perspectives of our project.
