http://2013.igem.org/wiki/index.php?title=Special:Contributions/LaurensDeBacker&feed=atom&limit=50&target=LaurensDeBacker&year=&month=2013.igem.org - User contributions [en]2024-03-28T14:12:31ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:KU_Leuven/Human_Outreach/EducationTeam:KU Leuven/Human Outreach/Education2013-10-05T03:13:39Z<p>LaurensDeBacker: </p>
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Our goal was to spark the interest of high school students in science and synthetic biology, and to tell them more about our project and the applications of synthetic biology. We have already visited several high schools in Flanders and we will visit some more after the Jamboree. <br/><br/><br />
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Schools visited so far: <br/><br />
17th of September: Sint-Dimpnacollege, GEEL <br/><br />
23th of September: Sint-Lodewijkscollege, BRUGGE <br/><br />
1st of October: European School of Mol, MOL <br/><br />
15th of October: Campus Sint-Aloysois, ZEPPEREN <br/><br />
17th of October: Sint-Jan Berchmanscollege, DIEST <br/><br />
22th of October: Sint-Ritacollege, KONTICH<br />
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<h3>Presentation</h3><br />
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The workshop we prepared consists of a presentation, some exercises with 'LegoBricks' we designed and an experiment at the end. You can find the presentation in <a href="https://static.igem.org/mediawiki/2013/8/88/Presentatie_iGEM%40School.pdf">Dutch</a> and in <a href="https://static.igem.org/mediawiki/2013/7/71/Presentation_iGEM%40School.pdf"> English</a> and our <a href="https://static.igem.org/mediawiki/2013/5/59/Tekst_iGEM%40School.pdf">text</a> in Dutch. <br />
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We started by giving a short introduction about DNA and its biochemistry. In general the students already had an introduction to DNA in class, but it was still very new to them. We then informed them about bacteria and which bacteria we use in the lab. We made a <b>3D-bacterial model</b>, which gave the students an idea of what a bacterium looks like. The model was printed by Fablab and consists of 3 layers. Inside, there is a representation of the cytoplasm, the plasmid and the bacterial genome.<br/><br />
After the introduction we came to the main part of our workshop: explaining what synthetic biology and its applications are. As an example, we discussed the development of modified yeast that can produce artemisinin to treat malaria. Subsequently, we presented our project by illustrating the mechanisms and properties of our bacteria. Our last topic in the presentation was the discussion about the ethical and social implications of synthetic biology in general, as well as in our project. <br />
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After the presentation, we gave the students <b> two exercises about building new systems with plastic puzzle pieces</b>. We made promotors, genes, regulators and proteins out of plexiglas in different colours. <br/><br />
First they had to make a biosensor system developed by the iGEM team of Groningen in 2009. The biosensor can detect arsenic and the bacteria can absorb this arsenic out of the water. To do this, they used an arsenic inducible promotor, followed by a gene encoding for a protein that helps with the production of hydrogen. <br/> <br />
In the second exercise, we used our ED Frosti model circuit (iGEM project of KU Leuven 2011). ED Frosti can defrost ice and freeze water. We made a simplified system of ED Frosti, consisting of two activators, three promotors, four genes and a feedback-loop.<br />
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<h3>Experiment</h3><br />
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The experiment proves that gene expression can be inducible so bacteria can adapt to different circumstances. If there’s lactose in the environment, <i>E. coli</i> will produce β-galactosidase to hydrolyse the lactose. When there’s no lactose, β-galactosidase won’t be produced. We didn’t use lactose, but we used ONPG. ONPG is a substrate of β-galactosidase and will turn yellow after hydrolysis. The yellow colour is proof of the presence of β-galactosidase. We couldn’t use <i>E. coli</i> in the high-school because of the safety regulations, but we explained the principle of inducible genes and showed the enzymatic reaction with β-galactosidase and ONPG. The students did this experiment themselves and they worked with micropipettes for the first time. <br />
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<h3>Feedback from the schools</h3><br />
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On the 17th of September, Flore and Sabine went to the Sint-Dimpnacollege. It was the first time we gave the workshops and everything went well. We gave the workshop three times to students in the sixth year of science-mathematics and latin-science. The students were enthusiastic and very interested. We got <a href="http://www.sdc.kogeka.be/nieuws/510/voorstelling-igem-project-door-studenten-ku-leuven">positive feedback</a> from the teachers and students.<br/><br />
On the 23th of September, Lukas and Bert went to the Sint-Lodewijkscollege. These students were also very enthusiastic and interested in synthetic biology and our project. We got a lot of good responses to our workshop. <br/><br />
We also went to a European school. Aurelie and Lukas gave the workshop to a selection of international students. The students were from the Dutch, English and French section. Here we gave the presentation in English. The teachers here were so impressed with our Plexiglas biobrick system that they asked if they could buy it from us (maybe entrepeneurial iGEM competition next year?)!<br />
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</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/qPCRTeam:KU Leuven/Project/Glucosemodel/qPCR2013-10-05T03:09:02Z<p>LaurensDeBacker: </p>
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<h3>Why qPCR?</h3><br />
<p align="justify">We performed this qPCR for two main reasons:</p><br />
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<ol><li>With a qPCR we can check if our genes of interest are properly transcribed. This is a good characterisation of the methyl salicylate brick <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060003">(BBa_K1060003)</a>.</li><br />
<li>We would like to know the amount of transcripts as an input for our <a href="https://2013.igem.org/Team:KU_Leuven/Project/MeSa/modeling">methylsalicylate model</a>. Because the mRNA production step is responsible for a large part of uncertainty in our model we would like to bypass it. In order to attain our goal without needing the transcription rates we tried to determine the <i>in vivo</i> mRNA concentrations using qPCR.</li></ol><br />
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The notebook on the qPCR experiments can be found in the <a href="https://2013.igem.org/Team:KU_Leuven/Journal/MeS/qPCR">journal</a>.<br />
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<h3>Sample Preparation</h3><br />
<p align="justify">We first started with the sample preparation, as described in the <a href="https://2013.igem.org/Team:KU_Leuven/Protocols#qRT-PCR_Protocol"> qPCR protocol </a>. For this experiment we used our regular <i>E. coli</i> strains (DH5α), harbouring our <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060003">methyl salicylate brick</a> and used 3 biological repeats.<br/><br />
After preparing three samples we started with the isolation of RNA. In a next step we removed DNA contamination by doing an extra DNase (Turbo DNase, Ambion) step as described in our protocol. <br/><br />
We then used the Nanodrop to get to know the concentration of our RNA and got the following results:</p><br />
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<tr><th></th><th>ng/µl</th><th>260/280</th><th>260/230</th></tr><br />
<tr><td>Sample A</td><td>100,3</td><td>2,19</td><td>1,42</td></tr><br />
<tr><td>Sample B</td><td>56,3</td><td>2,17</td><td>0,78</td></tr><br />
<tr><td>Sample C</td><td>92</td><td>2,17</td><td>1,28</td></tr><br />
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<p align="justify">These results are satisfactory. Only the 260/230 values are low because of the high salt level of the DNase buffer. After purification these values will rise.<br/></p><br />
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<h3>Genomic DNA contamination</h3><br />
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After the DNase step we needed to test whether there still is any DNA contamination in our RNA prep. We definitely have to remove all DNA contamination because the qPCR reaction will not differentiate between the original copy (gene on the plasmid) and the cDNA (Reverse Transcribed from the mRNA). If the original copy is still present in the sample, this will lead to overestimation of the quantity of mRNA molecules, and thus give false results. <br/><br />
To test whether there still is any DNA contamination we used PCR. We first tested if there is still any genomic DNA present in our sample by choosing a primer set on a conserved region. In our case we chose the csrA gene. As a positive control we used genomic DNA from a MG1655 strain. After gelelectrophoresis we got the following results. <br/><br />
We can conclude that we removed the genomic DNA successful since there are no visible bands for A, B and C (except for the primer dimer) (See Figure 1).<br/><br />
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<p align="justify">Figure 1 | DNA gel electrophoresis after a PCR that amplifies a genomic region (csrA). </p><br />
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<h3>Plasmid DNA contamination</h3><br />
<p align="justify">Since our genes of interest are not in the genome of our bacteria but on a plasmid, we have to check if all the plasmid DNA is completely removed. So we did the same PCR but with a primer set (iGEM0001 and iGEM0020) that amplifies a region on our plasmid. <br/><br />
We clearly still have plasmid DNA contamination even after a double DNase treatment. Which surprised us, because doing a double DNase treatment should have removed all of the contaminating DNA.<br />
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<p align="justify">Figure 2 | DNA gel electrophoresis after a PCR that amplifies a region on our plasmid.</p><br />
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<h3>Troubleshooting</h3><br />
<p align="justify">Thanks to some brainwork we got to a hypothesis that could explain this problem. Because we used a high copy number plasmid (ORI: pMB1), the proportion of plasmid DNA is far higher than the proportion of genomic DNA. The manufacturer advised to use a more rigorous treatment, but could not assure us that all traces of plasmid DNA will be removed because we are using a high copy number plasmid. So we had to find another way around of this problem.<br/><br />
After advice from dr. Basak Ozturk we decided to go for a restriction digest on the RNA samples. We figured that we’d best cut it with an enzyme that cuts the fragments that will be amplified in the qPCR itself. On this way the contaminating DNA will not be amplified while the cDNA will be correctly amplified and we could take unbiased conclusions.So we went looking for a good restriction enzyme that does the job perfectly for our two genes of interest.</p><br />
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<p align="justify">We came to the restriction enzyme <i>MvaI</i>. This should cut our plasmid on the right places as shown on figure 3.<br/><br />
The <i>MvaI</i> restriction enzyme should cut inside both of the qPCR amplified regions (the regions between the “blue boxes”), so that only linear amplification is possible there and that interference should be eliminated.<br/><br />
Unfortunately, after running some PCR’s and DNA electrophoresis gels to check whether we finally got rid of our contaminating DNA, we still had a disappointing result (see figure 4).<br/><br />
It might not be clear on the images but all our samples still show bands which means that our digestion-trick did not work. <br/><br />
This means that we are unable to remove the contaminating plasmid DNA. As previously mentioned we think that is due to the fact that the proportion of plasmid DNA is far higher than the proportion of genomic DNA, as we used a high copy number plasmid. This higher proportion of plasmid DNA is very hard to remove using DNase treatment. If we really want to go on with the qPCR, the only option is to clone our genes of interest in a low copy number vector or into the genome of our host strain. Since we were already quite busy with cloning, we decided to end the qPCR adventure here.</p><br />
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<p align="justify">Figure 4 | Drawing that shows the MvaI restriction sites. The blue boxes show the primers we designed for the qPCR. </p><br />
<img src="https://static.igem.org/mediawiki/2013/7/72/Control_PCR_RNA_samples_after_MvaI.jpg" alt="ControlPCR"/><br />
<p align="justify">Figure 5 | DNA gel electrophoresis after two different PCR reactions where each amplifies a different region on our plasmid.</p><br />
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<h3>Conclusion</h3><br />
<p align="justify">We decided to publish this story (even though it was unsuccesful) to warn the other teams for the difficulties that come with the removal of contaminating plasmid DNA in a RNA sample preparation.</p><br />
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Our goal was to spark the interest of high school students in science and synthetic biology, and to tell them more about our project and the applications of synthetic biology. We have already visited several high schools in Flanders and we will visit some more after the Jamboree. <br/><br />
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Schools visited so far: <br/><br />
17th of September: Sint-Dimpnacollege, GEEL <br/><br />
23th of September: Sint-Lodewijkscollege, BRUGGE <br/><br />
1st of October: European School of Mol, MOL <br/><br />
15th of October: Campus Sint-Aloysois, ZEPPEREN <br/><br />
17th of October: Sint-Jan Berchmanscollege, DIEST <br/><br />
22th of October: Sint-Ritacollege, KONTICH<br />
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<h3>Presentation</h3><br />
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The workshop we prepared consists of a presentation, some exercises with 'LegoBricks' we designed and an experiment at the end. You can find the presentation in <a href="https://static.igem.org/mediawiki/2013/8/88/Presentatie_iGEM%40School.pdf">Dutch</a> and in <a href="https://static.igem.org/mediawiki/2013/7/71/Presentation_iGEM%40School.pdf"> English</a> and our <a href="https://static.igem.org/mediawiki/2013/5/59/Tekst_iGEM%40School.pdf">text</a> in Dutch. <br />
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We started by giving a short introduction about DNA and its biochemistry. In general the students already had an introduction to DNA in class, but it was still very new to them. We then informed them about bacteria and which bacteria we use in the lab. We made a <b>3D-bacterial model</b>, which gave the students an idea of what a bacterium looks like. The model was printed by Fablab and consists of 3 layers. Inside, there is a representation of the cytoplasm, the plasmid and the bacterial genome.<br/><br />
After the introduction we came to the main part of our workshop: explaining what synthetic biology and its applications are. As an example, we discussed the development of modified yeast that can produce artemisinin to treat malaria. Subsequently, we presented our project by illustrating the mechanisms and properties of our bacteria. Our last topic in the presentation was the discussion about the ethical and social implications of synthetic biology in general, as well as in our project. <br />
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After the presentation, we gave the students <b> two exercises about building new systems with plastic puzzle pieces</b>. We made promotors, genes, regulators and proteins out of plexiglas in different colours. <br/><br />
First they had to make a biosensor system developed by the iGEM team of Groningen in 2009. The biosensor can detect arsenic and the bacteria can absorb this arsenic out of the water. To do this, they used an arsenic inducible promotor, followed by a gene encoding for a protein that helps with the production of hydrogen. <br/> <br />
In the second exercise, we used our ED Frosti model circuit (iGEM project of KU Leuven 2011). ED Frosti can defrost ice and freeze water. We made a simplified system of ED Frosti, consisting of two activators, three promotors, four genes and a feedback-loop.<br />
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<h3>Experiment</h3><br />
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The experiment proves that gene expression can be inducible so bacteria can adapt to different circumstances. If there’s lactose in the environment, <i>E. coli</i> will produce β-galactosidase to hydrolyse the lactose. When there’s no lactose, β-galactosidase won’t be produced. We didn’t use lactose, but we used ONPG. ONPG is a substrate of β-galactosidase and will turn yellow after hydrolysis. The yellow colour is proof of the presence of β-galactosidase. We couldn’t use <i>E. coli</i> in the high-school because of the safety regulations, but we explained the principle of inducible genes and showed the enzymatic reaction with β-galactosidase and ONPG. The students did this experiment themselves and they worked with micropipettes for the first time. <br />
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<h3>Feedback from the schools</h3><br />
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On the 17th of September, Flore and Sabine went to the Sint-Dimpnacollege. It was the first time we gave the workshops and everything went well. We gave the workshop three times to students in the sixth year of science-mathematics and latin-science. The students were enthusiastic and very interested. We got <a href="http://www.sdc.kogeka.be/nieuws/510/voorstelling-igem-project-door-studenten-ku-leuven">positive feedback</a> from the teachers and students.<br/><br />
On the 23th of September, Lukas and Bert went to the Sint-Lodewijkscollege. These students were also very enthusiastic and interested in synthetic biology and our project. We got a lot of good responses to our workshop. <br/><br />
We also went to a European school. Aurelie and Lukas gave the workshop to a selection of international students. The students were from the Dutch, English and French section. Here we gave the presentation in English and they were also very enthusiastic. <br />
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</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeSTeam:KU Leuven/Project/Glucosemodel/MeS2013-10-05T02:35:38Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">Methyl Salicylate</h3><br />
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<p align="justify">Methyl salicylate is an organic ester also known as <b>wintergreen oil</b>. It is produced by different species of plants. It is converted from <b>chorismate with salicylate as an intermediate</b>. In <i>E. coli</i>, chorismate is used as a precursor for the amino acids phenylalanine, tryptophane and tyrosine. It is produced from erythrose-4-phosphate (Ery4P) and phosphoenolpyruvate (PEP) through the shikimate pathway (Sprenger <i>et al.</i>, 2007). Chorismate is also the precursor of isochorismate, which is used for the biosythesis of quinones, siderophores and folic acid (Dosselaere and Vanderleyden, 2001).</p><br />
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<p align="justify">Figure 1 | Methyl Salicylate</p><br />
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<h3 class="bg-green">The Pathway To Methyl Salicylate</h3><br />
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<img src="https://static.igem.org/mediawiki/2013/e/ec/Shikimate_pathway.png" alt="shikimate pathway"/><br />
<p align="justify">Figure 2 | The Shikimate Pathway</p><br />
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<p align="justify">The conversion of Ery4P and PEP to DAHP (3-deoxy-D-arabinoheptulosonate 7-phosphate) is catalysed by DAHP synthase, an enzyme which exists in three isoforms, coded by the genes <i>aroF, aroG</i> and <i>aroH</i>. These isoforms are selectively inhibited by tyrosine, phenylalanine and tryptophan, respectively. In <i>E. coli</i>, about 80%, 20% and 1% of the enzyme activities are contributed by the DAHPS phenotypes of the <i>aroG, aroF</i> and <i>aroH</i> products, respectively (Ikeda <i>et al.</i>, 2006).<br/><br />
This means that when there is <b>enough phenylalanine</b> produced by the cell, or enough present in the medium, the <b>production of DAHP, and hence chorismate, will stop by inhibition of the <i>aro</i> genes</b>. The <b>production of phenylalanine will stop as well by inhibition of <i>pheA</i>,</b> coding for chorismate mutase/prephenate dehydratase. <br/><br />
The conversion from chorismate to methyl salicylate starts with a conversion to isochorismate, catalysed by the enzyme isochorismate synthase, encoded by the <i>pchA</i> gene. This isochorismate is directly converted to salicylate by isochorismate pyruvate/lyase, encoded by the <i>pchB</i> gene. This conversion happens immediately, since the <i>pchA</i> and <i>pchB</i> gene form the <i>pchBA</i> operon which is always transcribed to one mRNA. Conversion of salicylate to methyl salicylate is catalysed by S-adenosylmethionine-dependent methyltransferase, encoded by <i>bsmt1</i> (Gaille <i>et al.</i>, 2003). <br />
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<h3>A Biobrick to start from</h3><br />
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<p align="justify">The <b>iGEM team of MIT 2006 already constructed a Biobrick </b>(<a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a>) encoding the <i>pchBA</i> and <i>bsmt1</i> genes, so introducing this brick should induce the production of methyl salicylate, since chorismate is a common metabolite in <i>E. coli</i>. The MIT team discovered however that there was almost no methyl salicylate production observed, only when salicylic acid was added to the medium. <b>Our experiments with this Biobrick confirmed this lack of methyl salicylate production </b>(see the smell test in the MeS journal). There had to be something wrong with the conversion of chorismate to salicylate. One possibility is that there is something wrong with the enzymes produced by the <i>pchBA</i> genes. Another one is that there is a lack of chorismate present in the cell. We have attempted to increase the productivity of the MIT 2006 brick by using stronger ribosome binding sites. Also, <b>we replaced the <i>lac</i> promoter by a TetR-repressible promoter, so the lacI produced in our system doesn't interfere with the transcription of <i>pchBA</i>.</b>.<br />
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<img src="https://static.igem.org/mediawiki/2013/5/5c/Mesabiobrick.png" alt="mesa biobrick"/><br />
<p align="justify">Figure 3 | The original methyl salicylate biobrick <a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a></p><br />
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<p align="justify">Since chorismate is the precursor for three amino acids in <i>E. coli</i>, we believe that there is not much of the chorismate left to be converted to methyl salicylate, since the cell needs to keep producing a steady amount of essential amino acids.<br/><br />
To overcome this problem, we looked at a study by Sun <i>et al.</i> (2011), in which a synthetic pathway was introduced for the production of mandelic acid from chorismate. They achieved this by deleting different genes encoding enzymes that catalyse competing pathways, as well as by introducing a feedback-insensitive DAHP synthase mutant to increase the carbon flow down the shikimate pathway. This last method gave us inspiration to overcome our own problem.<br/><br />
If we look at the pathway in Figure 2, we can conclude that if the amino acids are present in the medium, the conversion of chorismate to these amino acids will be inhibited allosterically, as wel as the production of DAHP. <b>Our plan is to mutate the <i>aroG</i> gene in a manner that the enzymatic function still remains, but that it is insensitive to allosteric inhibition by phenylalanine. We only mutate the <i>aroG</i> gene since this isoform is responsible for 80% of DAHP synthase activity </b> (Hu <i>et al.</i>, 2003).</p><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/ec/Mutations.png"></center><br />
<p align="center">Figure 4 | The different pointmutations of <i>aroG</i> and their effect</p><br />
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<h3>Necessary Mutations</h3><br />
<p align="justify">It is proven that a Pro150Leu point mutation is Phe-insensitive. This mutation is used as a positive control in a study by Hu <i>et al.</i> (2003) in which they compare the effects of different mutation in the <i>aroG</i> gene on the specific enzymatic activity. The results showed that a Leu175Asp mutation also lead to a Phe-insensitive enzyme. Leu175 is located at the bottom of the possible inhibitor binding pocket, and it is believed to be a critical residue.<br/><br />
For reasons still unknown, L175D mutation showed an increased specific enzymatic activity compared to the wild type. In more recent studies, L175D is mostly used to obtain a Phe-insensitive DAHPS (Lin <i>et al.</i>, 2012). That is why we will try to <b>introduce a plasmid containing a L175D mutated <i>aroG</i> gene</b>.<br/><br />
It is also proven that transcription of the normal DAHP synthase gene will be inhibited when phenylalanine is present in the medium, so only the mutated form will be produced (Adhja & Gottesman, 1984).<br />
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<p align="justify">To functionally test our Methyl salicylate production brick (<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a>), we prepared 68 microcentrifuge tubes with <i>E.coli</I> expressing the MeS construct to conduct a blind smell test. This experiment consisted of 4 sets, different in incubation time and temperature. The first set was grown at room temperature for 24 hours, the second was incubated at 37ºC for 24 hours, the third was grown at 37ºC for 8 hours and the last set was grown at room temperature for 8 hours. In each set, we inoculated our brick <a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a> with IPTG, IPTG + salicylate (concentration range from 0.01mM to 1mM), and IPTG + chorismate (concentration range from 0.01mM to 1mM), the same for MIT 2006 brick <a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_J45700</a>. In addition, the same combinations of additives were introduced in parallel for our negative controls (non-MeS producing <i>E. Coli</i>). <br/><br />
We asked 11 people to participate in this blind test and calculated the percentage of people who smelled MeS. We can conclude that <b>the MeS/wintergreen smell was stronger after 24 hours incubation time compared to 8 hours. Growing the bacteria at 37 degrees also produced a more pronounced smell than those at room temperature. What’s more, in the microcentrifuge tubes where 0.1mM or 1mM salicylate was added, the smell became more obvious both after 8 and 24hrs of growth. The positive effect of adding chorismate became noticable after 24 hours</b>. The reason why chorismate requires more time to show an effect may be that chorismate is the very first precursor of the methyl salicylate production pathway, whereas the salicylate is an intermediate closer to the end product. As such, the impact of salicylate on the end product will become noticeable sooner. Most importantly, our brick worked in terms of producing methyl salicylate. <br/><br />
The results are shown below:</p><br />
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<p align="justify">Figure 8 | Incubate at room temperature for 8 hours</p><br />
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Lanes a-d were induced at OD600nm 0.5, Lanes e-h were induced at OD600nm 1.0 with the indicated concentration of IPTG.<br />
Cells were grown further for 1 hrs at 25°C. An identical experiment was performed with cells grown for 6hrs after induction. Finally, the brick was also tested with cells grown at 37°C.<br />
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Similar to our set-up for the EBF synthase protein expression, we tested the MIT biobrick (<a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a>) and our novel MeS construct (<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a>) via protein expression. We transformed our constructs in an <i>E.coli</i> expression strain, grew them at various temperatures (room temperature and 37 degrees celcius) and induced expression with increasing amounts of IPTG. We also added salicylate or chorismate to the growth medium in an attempt to increase MeS production.</br><br />
The figure shows results obtained with the BBa_J45700 brick. Our biobrick (BBa_K1060003) showed similar results (data not shown). Increasing the amount of IPTG did not influence the protein expression profile (compare lanes a-d or lanes e-h) but we do see some bands in the lanes a-d which we cannot see in lanes e-f (eg a band just above 55 kDa). Nonetheless, this observation can be verified with lower amounts of the protein extracts. Nonetheless, our smell test would suggest that the MeS brick does work. Hence, we need a more sensitive approach to identify the protein production of the MeS brick. Possible approaches would be via classic western blot experiment or a GC-MS set-up.<br />
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<b>We created the following BioBricks for the methyl salicylate part:</b><br />
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<a href="http://parts.igem.org/Part:BBa_K1060000" target="_blank">BBa_K1060000</a> coding biobrick of DAHP synthase, encoded by the <i>aroG</i> gene from <i>E. coli</i> in the pSB1C3 backbone. The insert is 1053 bp long.<br />
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<p align="justify" class="greytext"> Figure 9 | <i>aroG</i> coding sequence (1053 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with <i>EcoRI</i> and <i>PstI</i> restriction sites. Sequence confirmed. </p> <br />
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<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a> generator biobrick. It is a twin of the <a href="http://parts.igem.org/Part:BBa_J47500" target="_blank">BBa_J47500</a> biobrick made by the 2006 MIT team, put into a pSB1C3 backbone. The coding sequence is 3255 bp long.<br />
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<p align="justify" class="greytext">Figure 10 | Methyl salicylate producing construct (3255 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with <i>EcoRI</i> and <i>PstI</i> restriction sites. Sequence confirmed. </p><br />
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<a href="http://parts.igem.org/Part:BBa_K1060004" target="_blank">BBa_K1060004</a> intermediate biobrick consisting of <i>bsmt1</i> followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1177 bp long. Sequence confirmed.<br />
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<a href="http://parts.igem.org/Part:BBa_K1060005" target="_blank">BBa_K1060005</a> intermediate biobrick consisting of <i>pchBA</i> followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1839 bp long. Sequence confirmed.</p><br />
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Dosselaere, F., Vanderleyden, J., A metabolic node in action: chorismate-utilizing enzymes in microorganisms, Crit Rev Microbiol 2001;27(2):75-131.<br/><br />
Gaille, C., Reimmann, C., Haas D., Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of <i>Pseudomonas aeruginosa</i>, The Journal of Biological Chemistry 2003, 278(19):16893-16898.<br/>Hu, C., Jiang, P., Xu, J., Wu, Y., Huang, W., Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of <i>Escherichia coli</i>, J. Basic Microbiol. 43 (2003) 5, 399-406.<br/><br />
Ikeda, M., Towards bacterial strains overproducing L-tryptophan and other aromatics by metabolic engineering, Appl Microbiol Biotechnol 2006, 69:615-626.<br/><br />
Lin, S., Meng, ., Jiang, J., Pang, D., Jones, G., OuYang, H. Ren, L., Site-directed mutagenesis and over expression of aroG gene of <i>Escherichia coli</i> K-12, International Journal of Biological Macromolecules 51 (2012) 915-919.<br/><br />
Sprenger, G., From scratch to value: engineering <i>Escherichia coli</i> wild type cells to the production of L-phenylalanine and other fine chemicals derived from chorismate, Appl Microbiol Biotechnol 2007, 75:739-749.<br/><br />
Sun, Z., Ning, Y., Liu, L., Liu, Y., Sun, B., Jiang, W., Yang, C., Yang, S., Metabolic engineering of the L-phenylalanine pathway in <i>Escherichia coli</i> for the production of S- or R-mandelic acid, Microbial Cell Factories 2011, 10:71.<br/><br />
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<p align="justify">Methyl salicylate is an organic ester also known as <b>wintergreen oil</b>. It is produced by different species of plants. It is converted from <b>chorismate with salicylate as an intermediate</b>. In <i>E. coli</i>, chorismate is used as a precursor for the amino acids phenylalanine, tryptophane and tyrosine. It is produced from erythrose-4-phosphate (Ery4P) and phosphoenolpyruvate (PEP) through the shikimate pathway (Sprenger <i>et al.</i>, 2007). Chorismate is also the precursor of isochorismate, which is used for the biosythesis of quinones, siderophores and folic acid (Dosselaere and Vanderleyden, 2001).</p><br />
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<p align="justify">Figure 1 | Methyl Salicylate</p><br />
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<img src="https://static.igem.org/mediawiki/2013/e/ec/Shikimate_pathway.png" alt="shikimate pathway"/><br />
<p align="justify">Figure 2 | The Shikimate Pathway</p><br />
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<p align="justify">The conversion of Ery4P and PEP to DAHP (3-deoxy-D-arabinoheptulosonate 7-phosphate) is catalysed by DAHP synthase, an enzyme which exists in three isoforms, coded by the genes <i>aroF, aroG</i> and <i>aroH</i>. These isoforms are selectively inhibited by tyrosine, phenylalanine and tryptophan, respectively. In <i>E. coli</i>, about 80%, 20% and 1% of the enzyme activities are contributed by the DAHPS phenotypes of the <i>aroG, aroF</i> and <i>aroH</i> products, respectively (Ikeda <i>et al.</i>, 2006).<br/><br />
This means that when there is <b>enough phenylalanine</b> produced by the cell, or enough present in the medium, the <b>production of DAHP, and hence chorismate, will stop by inhibition of the <i>aro</i> genes</b>. The <b>production of phenylalanine will stop as well by inhibition of <i>pheA</i>,</b> coding for chorismate mutase/prephenate dehydratase. <br/><br />
The conversion from chorismate to methyl salicylate starts with a conversion to isochorismate, catalysed by the enzyme isochorismate synthase, encoded by the <i>pchA</i> gene. This isochorismate is directly converted to salicylate by isochorismate pyruvate/lyase, encoded by the <i>pchB</i> gene. This conversion happens immediately, since the <i>pchA</i> and <i>pchB</i> gene form the <i>pchBA</i> operon which is always transcribed to one mRNA. Conversion of salicylate to methyl salicylate is catalysed by S-adenosylmethionine-dependent methyltransferase, encoded by <i>bsmt1</i> (Gaille <i>et al.</i>, 2003). <br />
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<h3>A Biobrick to start from</h3><br />
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<p align="justify">The <b>iGEM team of MIT 2006 already constructed a Biobrick </b>(<a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a>) encoding the <i>pchBA</i> and <i>bsmt1</i> genes, so introducing this brick should induce the production of methyl salicylate, since chorismate is a common metabolite in <i>E. coli</i>. The MIT team discovered however that there was almost no methyl salicylate production observed, only when salicylic acid was added to the medium. <b>Our experiments with this Biobrick confirmed this lack of methyl salicylate production </b>(see the smell test in the MeS journal). There had to be something wrong with the conversion of chorismate to salicylate. One possibility is that there is something wrong with the enzymes produced by the <i>pchBA</i> genes. Another one is that there is a lack of chorismate present in the cell. We have attempted to increase the productivity of the MIT 2006 brick by using stronger ribosome binding sites. Also, <b>we replaced the <i>lac</i> promoter by a TetR-repressible promoter, so the lacI produced in our system doesn't interfere with the transcription of <i>pchBA</i>.</b>.<br />
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<img src="https://static.igem.org/mediawiki/2013/5/5c/Mesabiobrick.png" alt="mesa biobrick"/><br />
<p align="justify">Figure 3 | The original methyl salicylate biobrick <a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a></p><br />
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<p align="justify">Since chorismate is the precursor for three amino acids in <i>E. coli</i>, we believe that there is not much of the chorismate left to be converted to methyl salicylate, since the cell needs to keep producing a steady amount of essential amino acids.<br/><br />
To overcome this problem, we looked at a study by Sun <i>et al.</i> (2011), in which a synthetic pathway was introduced for the production of mandelic acid from chorismate. They achieved this by deleting different genes encoding enzymes that catalyse competing pathways, as well as by introducing a feedback-insensitive DAHP synthase mutant to increase the carbon flow down the shikimate pathway. This last method gave us inspiration to overcome our own problem.<br/><br />
If we look at the pathway in Figure 2, we can conclude that if the amino acids are present in the medium, the conversion of chorismate to these amino acids will be inhibited allosterically, as wel as the production of DAHP. <b>Our plan is to mutate the <i>aroG</i> gene in a manner that the enzymatic function still remains, but that it is insensitive to allosteric inhibition by phenylalanine. We only mutate the <i>aroG</i> gene since this isoform is responsible for 80% of DAHP synthase activity </b> (Hu <i>et al.</i>, 2003).</p><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/ec/Mutations.png"></center><br />
<p align="center">Figure 4 | The different pointmutations of <i>aroG</i> and their effect</p><br />
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<p align="justify">It is proven that a Pro150Leu point mutation is Phe-insensitive. This mutation is used as a positive control in a study by Hu <i>et al.</i> (2003) in which they compare the effects of different mutation in the <i>aroG</i> gene on the specific enzymatic activity. The results showed that a Leu175Asp mutation also lead to a Phe-insensitive enzyme. Leu175 is located at the bottom of the possible inhibitor binding pocket, and it is believed to be a critical residue.<br/><br />
For reasons still unknown, L175D mutation showed an increased specific enzymatic activity compared to the wild type. In more recent studies, L175D is mostly used to obtain a Phe-insensitive DAHPS (Lin <i>et al.</i>, 2012). That is why we will try to <b>introduce a plasmid containing a L175D mutated <i>aroG</i> gene</b>.<br/><br />
It is also proven that transcription of the normal DAHP synthase gene will be inhibited when phenylalanine is present in the medium, so only the mutated form will be produced (Adhja & Gottesman, 1984).<br />
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<p align="justify">To functionally test our Methyl salicylate production brick (<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a>), we prepared 68 microcentrifuge tubes with <i>E.coli</I> expressing the MeS construct to conduct a blind smell test. This experiment consisted of 4 sets, different in incubation time and temperature. The first set was grown at room temperature for 24 hours, the second was incubated at 37ºC for 24 hours, the third was grown at 37ºC for 8 hours and the last set was grown at room temperature for 8 hours. In each set, we inoculated our brick <a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a> with IPTG, IPTG + salicylate (concentration range from 0.01mM to 1mM), and IPTG + chorismate (concentration range from 0.01mM to 1mM), the same for MIT 2006 brick <a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_J45700</a>. In addition, the same combinations of additives were introduced in parallel for our negative controls (non-MeS producing <i>E. Coli</i>). <br/><br />
We asked 11 people to participate in this blind test and calculated the percentage of people who smelt MeS. We can conclude that <b>the MeS/wintergreen smell was stronger after 24 hours incubation time compared to 8 hours. Growing the bacteria at 37 degrees also produced a more pronounced smell than those at room temperature. What’s more, in the microcentrifuge tubes where 0.1mM or 1mM salicylate was added, the smell became more obvious both after 8 and 24hrs of growth. The positive effect of adding chorismate became noticable after 24 hours</b>. The reason why chorismate requires more time to show an effect may be that chorismate is the very first precursor of the methyl salicylate production pathway, whereas the salicylate is an intermediate closer to the end product. As such, the impact of salicylate on the endproduct will become noticeable sooner. Most importantly, our brick worked in terms of producing methyl salicylate. <br/><br />
The results are shown below:</p><br />
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<p align="justify">Figure 5 | Incubate at room temperature for 24 hours</p><br />
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<p align="justify">Figure 6 | Incubate at 37 degrees for 24 hours</p><br />
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<p align="justify">Figure 8 | Incubate at room temperature for 8 hours</p><br />
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Lanes a-d were induced at OD600nm 0.5, Lanes e-h were induced at OD600nm 1.0 with the indicated concentration of IPTG.<br />
Cells were grown further for 1 hrs at 25°C. An identical experiment was performed with cells grown for 6hrs after induction. Finally, the brick was also tested with cells grown at 37°C.<br />
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Similar to our set-up for the EBF synthase protein expression, we tested the MIT biobrick (<a href="http://parts.igem.org/Part:BBa_J45700" target="_blank">BBa_J45700</a>) and our novel MeS construct (<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a>) via protein expression. We transformed our constructs in an <i>E.coli</i> expression strain, grew them at various temperatures (room temperature and 37 degrees celcius) and induced expression with increasing amounts of IPTG. We also added salicylate or chorismate to the growth medium in an attempt to increase MeS production.</br><br />
The figure shows results obtained with the BBa_J45700 brick. Our biobrick (BBa_K1060003) showed similar results (data not shown). Increasing the amount of IPTG did not influence the protein expression profile (compare lanes a-d or lanes e-h) but we do see some bands in the lanes a-d which we cannot see in lanes e-f (eg a band just above 55 kDa). Nonetheless, this observation can be verified with lower amounts of the protein extracts. Nonetheless, our smell test would suggest that the MeS brick does work. Hence, we need a more sensitive approach to identify the protein production of the MeS brick. Possible approaches would be via classic western blot experiment or a GC-MS set-up.<br />
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<b>We created the following BioBricks for the methyl salicylate part:</b><br />
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<a href="http://parts.igem.org/Part:BBa_K1060000" target="_blank">BBa_K1060000</a> coding biobrick of DAHP synthase, encoded by the <i>aroG</i> gene from <i>E. coli</i> in the pSB1C3 backbone. The insert is 1053 bp long.<br />
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<p align="justify" class="greytext"> Figure 9 | <i>aroG</i> coding sequence (1053 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with <i>EcoRI</i> and <i>PstI</i> restriction sites. Sequence confirmed. </p> <br />
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<a href="http://parts.igem.org/Part:BBa_K1060003" target="_blank">BBa_K1060003</a> generator biobrick. It is a twin of the <a href="http://parts.igem.org/Part:BBa_J47500" target="_blank">BBa_J47500</a> biobrick made by the 2006 MIT team, put into a pSB1C3 backbone. The coding sequence is 3255 bp long.<br />
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<p align="justify" class="greytext">Figure 10 | Methyl salicylate producing construct (3255 bp) in pSB1C3 backbone (2070 bp). Digestion confirmation, cut with <i>EcoRI</i> and <i>PstI</i> restriction sites. Sequence confirmed. </p><br />
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<a href="http://parts.igem.org/Part:BBa_K1060004" target="_blank">BBa_K1060004</a> intermediate biobrick consisting of <i>bsmt1</i> followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1177 bp long. Sequence confirmed.<br />
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<a href="http://parts.igem.org/Part:BBa_K1060005" target="_blank">BBa_K1060005</a> intermediate biobrick consisting of <i>pchBA</i> followed by a double terminator. It is in the pSB1C3 backbone and the insert is 1839 bp long. Sequence confirmed.</p><br />
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<h3 class="bg-green">References</h3><br />
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Dosselaere, F., Vanderleyden, J., A metabolic node in action: chorismate-utilizing enzymes in microorganisms, Crit Rev Microbiol 2001;27(2):75-131.<br/><br />
Gaille, C., Reimmann, C., Haas D., Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of <i>Pseudomonas aeruginosa</i>, The Journal of Biological Chemistry 2003, 278(19):16893-16898.<br/>Hu, C., Jiang, P., Xu, J., Wu, Y., Huang, W., Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of <i>Escherichia coli</i>, J. Basic Microbiol. 43 (2003) 5, 399-406.<br/><br />
Ikeda, M., Towards bacterial strains overproducing L-tryptophan and other aromatics by metabolic engineering, Appl Microbiol Biotechnol 2006, 69:615-626.<br/><br />
Lin, S., Meng, ., Jiang, J., Pang, D., Jones, G., OuYang, H. Ren, L., Site-directed mutagenesis and over expression of aroG gene of <i>Escherichia coli</i> K-12, International Journal of Biological Macromolecules 51 (2012) 915-919.<br/><br />
Sprenger, G., From scratch to value: engineering <i>Escherichia coli</i> wild type cells to the production of L-phenylalanine and other fine chemicals derived from chorismate, Appl Microbiol Biotechnol 2007, 75:739-749.<br/><br />
Sun, Z., Ning, Y., Liu, L., Liu, Y., Sun, B., Jiang, W., Yang, C., Yang, S., Metabolic engineering of the L-phenylalanine pathway in <i>Escherichia coli</i> for the production of S- or R-mandelic acid, Microbial Cell Factories 2011, 10:71.<br/><br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:39:28Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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<h3>gBlocks</h3><br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves versus the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we show the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:36:22Z<p>LaurensDeBacker: </p>
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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<h3>gBlocks</h3><br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves versus the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we show the most interesting results. The figure shows some slight additional bands in lane a (around 110 kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:35:30Z<p>LaurensDeBacker: </p>
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves versus the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we show the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:34:30Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves versus the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:32:39Z<p>LaurensDeBacker: </p>
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Reaction.jpg" alt="reaction"/></center><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves versus the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:28:49Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Reaction.jpg" alt="reaction"/></center><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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<h3>gBlocks</h3><br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<h3 class="bg-green">Our Bricks</h3><br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future, for example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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<h3 class="bg-green">The pathway to E-β-Farnesene</h3><br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:25:54Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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<h3>gBlocks</h3><br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the resulting video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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<h3 class="bg-green">The pathway to E-β-Farnesene</h3><br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:22:29Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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<h3 class="bg-green">General Background of the EBF synthase</h3><br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbone and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:19:51Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details on the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:14:45Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used plasmid pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:09:14Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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In the case of the EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site was present, which would conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:06:06Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch the transcription of the EBF synthase gene on and off.</p><br />
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site is present, which will conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:04:18Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of biomimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium</b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood)</b> and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch on/off the transcription of EBF synthase gene.</p><br />
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<h3>Gettin' the gene</h3><br />
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site is present, which will conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/Part:BBa_K1060008" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T01:01:02Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator.</b> EBF is not only made by aphids but also by plants and other organisms in a form of bio-mimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium </b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood) </b>and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch on/off the transcription of EBF synthase gene.</p><br />
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site is present, which will conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K106000" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T00:59:52Z<p>LaurensDeBacker: </p>
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In this part, we will give some more information about the E-β-farnesene (EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators</b> (e.g. the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects: changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5 and 7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator.We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator. </b> EBF is not only made by aphids but also by plants and other organisms in a form of bio-mimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium </b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood) </b>and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch on/off the transcription of EBF synthase gene.</p><br />
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site is present, which will conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K106000" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify"><b>The genes involved in the mevalonate pathway</b><br/>(Martin <i>et al.</i>)</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/EBFTeam:KU Leuven/Project/Glucosemodel/EBF2013-10-05T00:53:38Z<p>LaurensDeBacker: </p>
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<h3 class="bg-green">E-β-Farnesene</h3><br />
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In this part, we will give some more information about the E-β-farnesene (aka EBF) part of the project. EBF is an <b>alarm pheromone</b>, released by almost all of the 4000 aphid species known thus far <b>in response to the presence of predators </b>(eg the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, <b>EBF can also cause long term effects : changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc.</b> Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/><br />
Hence, having our BanAphids produce EBF should help to <a href="#aphid experiments">repel aphids</a> from our plant of choice. In the following sections, we will give you a <a href="#background">general background of EBF synthase</a> followed by an overview of the <a href="#model">model and the genes</a>, the <a href="#wetlab">wetlab work</a> and the <a href="#bricks">biobricks</a> we built for the EBF part. Finally we have made some suggestion how to optimise the production of EBF in the future. For this we will take you on a tour through <a href="#pathway">the pathways that result in EBF</a> and the problems that arise with this. Of course we have added possible <a href="#problemssolutions">solutions to these problems</a>.<br />
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We cloned and expressed the EBF synthase gene in <i>E. coli</i>. This enzyme will break down (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/><br />
The enzyme prefers bivalent cations as cofactors; eg a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF synthase function. The ideal pH for EBF synthase will be between 5.5-7.</p><br />
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The EBF construct we designed consists of a <b>constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator.We used <a href="http://parts.igem.org/Part:BBa_B0015" target="_blank">BBa_B0015</a> for the double terminator. </b> EBF is not only made by aphids but also by plants and other organisms in a form of bio-mimicry. We obtained two different sources of the EBF gene. One gene originates from the <b>soil bacterium </b><i>Streptomyces coelicolor</i> (Centre of Microbial and Plant Genetics of KU Leuven). We chose this plant-residing bacterium because it would be a perfect chassis for the ultimate expression of EBF in our <i>E. coligy</i> system. The other EBF gene is from <b>the plant <i>Artemisia annua</i> (sweet wormwood) </b>and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K<sub>M</sub> for the <i>Artemisia annua</i> protein is calculated at 0.0021 mM, with a Kcat/K<sub>M</sub>=4.5 and a turnover number of 0.0095 s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 mM and the turnover number 0.019 s<sup>-1</sup>. <br/><br />
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Unfortunately, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the <i>Artemisia annua</i> gene and product. <br />
For our construct, our first choice was a medium strength promoter with medium RBS (<a href="http://parts.igem.org/Part:BBa_K608006" target="_blank">BBa_K608006</a>); we nonetheless also made the construct with a strong promoter and RBS. The <i>lac</i> operator in front of the EBF synthase gene will allow us to switch on/off the transcription of EBF synthase gene.</p><br />
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with a colony PCR. The EBF synthase gene from <i>Artemisia annua</i> was received in the pET28 vector from professor Brodelius (Kalmar University, Sweden). In this gene an additional <i>EcoRI</i> restriction site is present, which will conflict with the standard iGEM cloning work. Therefore <b>we removed this site via site directed mutagenesis</b> after transferring the gene into the iGEM pSB1C3 backbone. <br />
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<h3>Cutting and pasting</h3><br />
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Once we obtained the target gene (EBF) in the standard pSB1C3 backbone, we started our cloning work. We used a pSB1C3 with a promoter or terminator as chassis, cut this open and inserted the gene of interest. When ligating the insert in front of the double terminator, we cut the vector with <i>EcoRI</i> and <i>XbaI</i>, and the insert with <i>EcoRI</i> and <i>SpeI</i>. The promotor vector on the other hand is cut with <i>SpeI</i> and <i>PstI</i> restriction sites, and the insert is cut with <i>XbaI</i> and <i>PstI</i> restriction sites. This works because <i>SpeI</i> and <i>XbaI</i> are isoschizomers.<br/><br />
Ligations were performed in parallel in two different ways. In one setup we ligated for 20 minutes at 16 ℃, and in comparison, the second ligation of the same products was conducted at 16 ℃ overnight.<br/><br />
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p><br />
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<h3>Confirmation</h3><br />
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After we observed colonies the next day, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert was in the vector, this was followed up by digestion confirmation after the plasmid extraction. <b>Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation</b>.<br />
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Meanwhile, we also built the EBF construct with a <i>lac</i> operator between the promoter and gene, using the gBlock principle. We designed the gBlocks, assembled them and ligated the insert into pSB1C3 backbone. The colonies obtained also went through the three confirmation steps mentioned above before we were satisfied. <br />
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult <a href="https://2013.igem.org/Team:KU_Leuven/Journal/EBF/wetlab">our wetlab journal</a>.<br />
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<b>After we overcame a lot of difficulties, we finally made the following bricks at the end of the summer.</b></p><ol><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001" target="_blank">BBa_K1060001</a> This is a coding biobrick with the EBF synthase gene from <i>Streptomyces coelicolor</i> in pSB1C3 backbone and an insert length of 1386bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002" target="_blank">BBa_K1060002</a> This is another coding biobrick with EBF synthase gene from <i>Artemisia annua</i> in pSB1C3 backbone and an insert length of 1725bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K106000" target="_blank">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009" target="_blank">BBa_K1060009</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> in the pSB1C3 backbonethe and an insert length of 1924bp.</li><br />
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<li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011" target="_blank">BBa_K1060011</a> This is a generator biobrick with a medium constitutive expression of EBF synthase from <i>Artemisia annua</i> AND a <i>lac</i> operator after the promoter and an insert length of 1965bp.</li><br />
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With our EBF synthase constructs ready, we tested them with several aphid experiments.<br/><br />
Our pilot experiment tested the medium strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</b></a>. We placed aphids on a leaf in the middle of a huge petri dish, an EBF-producing bacterium plate on the left, a control on the right. In <b>the results video we observed that the general trend of aphid movement was away from the EBF-producing bacterium. These results suggest our EBF synthase producing bacteria seemed to work.</b> <br/><br />
Moreover, we also tried another set-up with our high strength EBF synthase producing brick <b><a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</b></a>. This time we connected the leaves that were on the EBF-producing bacteria plate with those on the control plate and with the leaf in the middle where the aphids resided. This facilitates movement of the aphids to other leaves. However, there was no significant difference between the amount of aphids on the control leaves vs the <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K106011</a> leaves. The lac operator in this construct may interfere with the production of EBF.</p><br />
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In addition, we also examined the aphid's behavior without a leaf as a starting point. We put 30 aphids in the middle of a huge petri dish, on the left side we placed a leaf with 10µl of EBF-producing bacteria and on the right side we placed a non-treated leaf as control. Thus, we offered the aphids the chance to go searching for food. After 2 hours we counted the number of aphids on the leaves, there were 4 aphids on the leaf where the EBF was produced and 6 aphids on the control leaf, the rest of aphids just walked randomly in the big petri dish. For lack of time, we could unfortunately not repeat this experiment.<br/><br />
<b>Our pilot experiments did indicate a trend in the right direction. Several aspects of the setup will be optimized in the future. For example the amount of bacteria, the strength of the promoter, the ventilation of the setup, the incubation time and the temperature, etc. The reason for this is that the concentration of EBF is essential to trigger the desired response in the aphids. Both too high and too low concentrations will lead to aphid insensitivity.</b> <br/><br />
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As another approach to prove our constructs (<a href="http://parts.igem.org/Part:BBa_K1060009" target="_blank">BBa_K1060009</a>, <a href="http://parts.igem.org/Part:BBa_K1060011" target="_blank">BBa_K1060011</a> and <a href="http://parts.igem.org/Part:BBa_K1060014" target="_blank">BBa_K1060014</a>), we transformed our different EBF synthase bricks in an <i>E.coli</i> expression strain, grew these up under different temperatures, times and, if possible, IPTG induction levels. Bacterial pellets were harvested and proteins extracted.<br/><br />
Here we showed the most interesting results. The figure shows some slight additional bands in lane a (around 110kDa and around 60 kDa), the protein extract from the lacI operator medium strength promoter construct. These bands are less clear in the medium and high strength promoter lane. The expected size of the EBF synthase protein is around 66kDa which could fit with the lower band. Gel extraction and Mass Spectrometry based identification will confirm if these bands represent the EBF synthase gene and possibly the increased production of a secondary protein. Interestingly, the lacI medium promoter construct did not influence aphid behaviour. Possibly the expression of EBF synthase is just too high, which would be equally inhibitory as a too low concentration. Other approaches to better identify the functionality of this construct would be via a gas chromatography analysis to directly measure the amounts of EBF produced.<br />
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β-farnesene is a terpenoid that is converted from farnesyl pyrophosphate (FPP) by the enzyme β-farnesene synthase (EC 4.2.3.47). <br/><br />
<b>FPP is the precursor of β-farnesene</b>, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/><br />
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most <b>prokaryotes use the non-mevalonate or DXP pathway</b>, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. <b>Eukaryotes, except for plants, exclusively use the mevalonate pathway</b>, producing IPP starting from acetyl-CoA. Plants use both pathways.</p><br />
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<p align="justify">On the left you can see the non-mevalonate pathway or DXP pathway, showing the conversion of pyruvate and glyceraldehyde-3-phosphate to the terpenoid precursor IPP and its isomer DMAPP.<br/><br />
Pyr = pyruvate, G3P = glyceraldehyde-3-phosphate, DXP = 1-deoxy-D-xylulose 5-phosphate, MEP = 2-C-methylerythritol 4-phosphate, CDP-ME = 4-phosphocytidyl-2-C-methylerythritol, CDP-MEP = 4-phosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, MEcPP = 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, HMB-PP = (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, DXS = DXP synthase, DXR = DXP reductase, CMS = CDP-ME synthase, CMK = CDP-ME kinase, MCS = MEcPP synthase, HDS = HMB-PP synthase, HDR = HMB-PP reductase</p><br />
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<p align="justify">FPP is an important precursor, used for the biosynthesis of lots and lots of compounds. Once we insert a plasmid containing the β-farnesene synthase gene, we may obtain only a very small amount of β-farnesene, since the precursor amount wasn't increased and there simply isn’t enough FPP available to produce the amount of β-farnesene to fully use the capacity of the EBF synthase enzyme we brought in.<br/><br />
A solution may be to co-transform plasmids to engineer a mevalonate pathway in <i>E. coli</i>, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile. This was demonstrated many times by J.D. Keasling in <i>S. cerervisiae</i>, while Martin <i>et al.</i>, (2003) implemented this mevalonate pathway in <i>E. coli</i>. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in <i>E. coli</i>. However, expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. This once again shows the need for a controlled production of the elements in this pathway; too much is equally detrimental as too little.</p><br />
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<p align="justify">Since there are <b>eight genes </b>responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into <b>two parts</b>. A first plasmid named <b>pMevT</b>, responsible for the conversion of acetyl-CoA to mevalonate, harboring the <i>atoB</i>, <i>HMGS</i> and <i>tHMGR</i> genes into a pBAD33 vector, and a second one named <b>pMBIS</b>, harboring the <i>ERG12</i>, <i>ERG8</i>, <i>MVD1</i>, <i>idi</i> and <i>ispA</i> genes into a pBBR1MCS-3 plasmid. Coexpression of these two operons in an <i>ispC</i> deficient <i>E. coli</i> strain produced the terpenes, even in the absence of mevalonate, indicating that the mevalonate pathway works. <b>A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields</b>.</p><br />
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<p align="justify">Even though we do not need a very high production of EBF it would be definitely better to optimise the pathway, by using the plasmids pMevT and pMBIS, described above. <b>Implementing them into our BanAphids along with the synthetic β-farnesene synthase gene could result into high yields of β-farnesene. This way the amount of EBF can be easily changed via the amount of bacteria used or the concentration of the cofactor Mg<sup>2+</sup></b>.<br/>Due to the short amount of time iGEM offered we did not yet started doing this, but this is definitely something future teams might look into.</p><br />
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<p align="justify">Kajiwara S., Fraser P., Kondo K., Misawa N., Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid synthesis in Escherichia coli, Biochem J. 324, 421-426 (1997).<br/><br />
Martin V., Pitera D., Withers S., Newman J., Keasling J., Engineering a mevalonate pathway in Escherichia coli for production of terpenoids, Nature Biotechnology 21(7), 796-802 (2003). <br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T23:58:24Z<p>LaurensDeBacker: </p>
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It is of course very important to stay safe. Here we have gathered information on how we stayed <a href="#general safety">safe in our lab</a>, what the <a href="#europe legislation">government rules</a> and the <a href="#KUL">rules of the KU Leuven</a> are. Finally, we also elaborate on how we could keep our <a href="#bacterial safety">bacterium from spreading</a> if it would eventually be commercialised and released into the <a href="#environmental safety">environment</a>. As well as which specific mechanisms have we implemented to keep our <a href="#auxotrophic">bacteria contained</a>, with for instance, a <a href="#kill switch">'kill switch'</a>. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning certain safety issues.</p><br />
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Safety forms were <b>approved</b> on September 29, 2013 by the iGEM Safety Committee.<br />
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We have covered the safety aspect of our project in many ways. Within synthetic biology there should be three main risks that need to be covered: exposure of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem.<br />
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In the past three months, we have added new branches into the pathways of <i>E. coli</i>. Our experiments were performed with <b>non-pathogenic <i>E. coli</i> strains</b> (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms demands extra safety conditions; physical measures have been taken to separate the organism from the surrounding environment and vice versa.<br/><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria (<b>BanAphids</b>) will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are naturally occurring substances. EBF repels aphids and MeS attracts ladybugs, one of the aphid's natural enemies. The low concentrations needed for these substances to be effective is not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with <b>acetylsalicylic acid (aspirin)</b> and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our <b>BanAphids</b> could cause allergic reactions (swelling, redness, ...), but serious reactions only occur in high concentrations and are very rare.<br/><br />
The effect of <b>BanAphids</b> on the environment is not trivial and thorough field experiments must definitely be conducted. The maximum production rate of a single bacterium needs to be determined so that this could be scaled up through a model. This way we would be able to estimate the concentration of EBF and MeS in the surroundings if a whole field would be occupied by <b>BanAphids</b>. We can then determine the necessary parameters in order to minimise ecological disruption according to these calculated estimates of surrounding EBF and MeS concentrations. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present, therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<p align = "justify">Research concerning genetically modified organisms (GMOs) is subjected to strict safety legislation in Belgium. These laws are in place to protect the general public and the environment from the potential risks of GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB) (section Biosafety and Biotechnology of the Scientific Institute of Public Health) which acts as the scientific secretary of the board. They evaluate the risk analysis in the licence request. At the University of Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
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The KU Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for <b>Health, Safety and Environment (HSE)</b> issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the KU Leuven. The safety training consisted of a summary of the important biosafety issues and the composition of the lab. At the beginning of our project, all members of the iGEM team have received an update from the KU Leuven's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br/><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we discussed the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof. Dr. Johan Robben and Dr. Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. They have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. For instance we talked to the company Perseus (Partner in Regulatory and Safety Services) to discuss the possible hurdles we would face and which safety aspects we need to incorporate. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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If our project would be used outside the laboratory, many parameters would need to be determined. As stated earlier, the pheromones produced by the <b>BanAphids</b> are not harmful for our health unless available in high concentrations. So if the <b>BanAphids</b> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
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In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested by Moe-Behrens <i>et al.</i>, 2013, containment through engineered auxotrophy so that <b>BanAphids</b> are unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We also believe that providing a bacterium with an advantage eg. antibiotic resistance, provides bacteria with the opportunity to mutate into even stronger bacteria. We therefore propose the use of a tryptophan auxotrophic bacterium.<br/><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition and we find it very important that the work of previous iGEM teams do not go to waste which is why we decided to look into several 'kill switches' developed by previous iGEM teams.</p><br />
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Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A, B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat (Moe-Behrens <i>et al.</i>, 2013).</p><br />
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We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our 'sticker' or 'oscillator' model. <br/><br />
The basic mechanism of the <b>tryptophan auxotrophic bacteria</b> is based on the fact that they are unable to synthesise the essential compound, tryptophan, required for their survival, which means we must provide tryptophan in their medium. They therefore rapidly die once they escape from the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produces a toxic protein that disrupts the bacterial cell's membrane and kills the cells. A stochastic activation of this toxic component could also be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
This is a good candidate for the biocontainment of <b>BanAphids</b> in the 'sticker' <a href="https://2012.igem.org/Team:Groningen/Sticker">(Design of Groningen iGEM 2012)</a> or 'oscillator' model. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape. However, we need a different mechanisms to contain the <b>BanAphids</b> of our 'spray' or 'glucose' model.</p><br />
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The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g. IPTG) is added, and this will activate a cascade mechanism that will kill the cells. This induced lethality could, for instance, be used to clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Furthermore, as suggested by Balan <i>et al.</i> (2005), the use of a nuclease enzyme suicide system disfavours horizontal gene transfer compared to the above suggested suicide system that targets the cell membrane. If a bacterial cell is lysed, that bacterium might be dead but other bacteria in the environment are not dead (yet) and a suicide system that targets the disruption of the cell membrane does not guarantee the degradation of DNA. The lysed bacterium's DNA is now free and available to be taken up by neighbouring bacteria. For this reason we have chosen a 'kill switch' that activates a nuclease system. <br/><br />
Inducible kill systems are not new to iGEM, there are already several constructs available in the database, such as the inducible <i>BamHI</i> system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, spontaneous genetic mutations in this lethal <i>BamHI</i> gene is a central cause of safeguard failure. Spontaneous genetic mutations can deactivate lethal gene expression causing failure of biocontainment. As these engineered cells propagate in the laboratory, or in large bioreactors, disfunctional genetic safeguards can gradually accumulate in the population. If this accumulation is high enough, then the synthetic organisms might survive in the environment after disposal or accidental release (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Calgary 2012 Team</a>.</p><br />
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Moe-Behrens, G. H. G., Davis, R., Haynes, K., Preparing synthetic biology for the world, Frontiers in Microbiology 2013, 4(5): 1-10. <br/><br />
Balan, A., Schenberg, A. C. G., A conditional suicide system for Saccaromyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease, Yeast 2005; 22; 203-212.<br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/DataPageTeam:KU Leuven/Project/DataPage2013-10-04T23:33:31Z<p>LaurensDeBacker: </p>
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Welcome to our data page! Here we will summarize everything we achieved this summer. Of course, if you want a more extensive explanation you should check out the corresponding wiki page.<br />
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<p align="justify">Ultimately our project aims to <b>reduce crop loss due to aphid infestations</b>. With an environmental project like ours, the computer is our best friend: through modelling and prediction algorithms we can reduce the real costs of field tests. Moreover, as iGEM team we were unable to conduct a field experiment for our BanAphids during this summer.<br/><br />
Our first step in the modelling was to <b>predict the effect of our pheromones on the environment and the ecosystem</b> through a series of modelling steps. You can find this in our <a href="https://2013.igem.org/Team:KU_Leuven/Project/Ecological/Modelling" target="_blank">ecological model page</a>. <br />
The pheromones E-β-farnesene and methyl salicylate will not only affect the environment but also the bacterial cell itself. To figure out the impact of our system on our <i>E. coli</i> we performed a <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS/Modelling-FBA" target="_blank">Flux Balance Analysis</a>. Once we knew that our bacteria could handle the production of the pheromones we tried to <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS/Modelling" target="_blank">predict the exact production amounts</a> and find the rate limiting steps. Here our goal was also to feed wet-lab data into our algorithms. Finally, to optimise the impact of the released pheromones on the aphids and the ladybugs, we designed an <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator/Modelling" target="_blank">oscillating transcription factor network</a> to regulate their production. This oscillator also communicates between cells, enforcing the oscillating rhythm onto the whole colony.<br/><br />
Summarized, <b>these algorithms allow us to model our system from the cellular metabolism throughout to the environmental impact</b>. Based on our models, we adapted the actual building of the system towards the most effective circuit. Finally, our mathematical predictions will provide significant benefits once we prepare our E. coligy for field tests. <br />
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<h3>Ecology</h3><br />
<p align="justify"><b>Two companies specialized in insects were very interested in our project. They invited us to perform experiments at their facilities.</b> These experiments proved that methyl salicylate not only attracts predators but also activates & protects the plant against aphids. They also proved that aphids are driven away by methyl salicylate.<br/><br />
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<p align="justify">As a result of our <b>unique collaboration between philosophers and scientists within our team, we formulated several new approaches to ethical and philosophical considerations</b>. The best example is the <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Descriptive" target="_blank"> descriptive evaluation</a> in which all team members and supervisors were interviewed about their ethical beliefs. This bottom-up collaboration between students is a new starting point for approaching human practices within the iGEM competition. We also explained why participants should inform the general public using the ideas of philosopher <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/HannahArendt" target="_blank">Hannah Arendt</a> and we examined the responsability of synthetic biologists using the ideas of philosopher <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/HansJonas" target="blank">Hans Jonas</a>. <b>This is not only a first time in the iGEM competition, but has also never been done in the scientific literature!</b><br/><br />
<p align="justify">To make sure that we have <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/EndUser" target="blank">a product that is worth to be launched</a> we asked Biobest & pcfruit why they were so interested in the BanAphids. <b>We also asked farmers whether they would use our BanAphids and the results were definitely positive!</b></p><br />
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<p align="justify"><b>We organized a symposium for the general public for which we also invited the other BeNeLux teams to present their project.</b> We hope that one day this will become a yearly tradition in the iGEM competition.<br/><br />
We went to schools to teach them about synthetic biology. For this we made our very own Plexiglas "biobricks" which the students can use to work on excercises and we made a <b>3D-bacterial model</b>, which gives the students an idea of what a bacterium looks like</p><br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/DataPageTeam:KU Leuven/Project/DataPage2013-10-04T23:30:37Z<p>LaurensDeBacker: </p>
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<p align="justify"><br />
Welcome to our data page! Here we will summarize everything we achieved this summer. Of course, if you want a more extensive explanation you should check out the corresponding wiki page.<br />
</p><br />
</div><br />
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<br />
<div id="header" class="row-fluid"><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align="justify">Ultimately our project aims to <b>reduce crop loss due to aphid infestations</b>. With environmental projects like ours, the computer is our best friend: through modelling and prediction algorithms we can reduce the real costs of field tests. Moreover, as iGEM team we were unable to conduct a field experiment for our BanAphids during this summer.<br/><br />
Our first step in the modelling was to <b>predict the effect of our pheromones on the environment and the ecosystem</b> through a series of modelling steps. You can find this in our <a href="https://2013.igem.org/Team:KU_Leuven/Project/Ecological/Modelling" target="_blank">ecological model page</a>. <br />
The pheromones E-β-farnesene and methyl salicylate will not only affect the environment but also the bacterial cell itself. To figure out the impact of our system on our E. coli we performed a <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS/Modelling-FBA" target="_blank">Flux Balance Analysis</a>. Once we knew that our bacteria could handle the production of the pheromones we tried to <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS/Modelling" target="_blank">predict the exact production amounts</a> and find the rate limiting steps. Here our goal was also to feed wet-lab data into our algorithms. Finally, to optimise the impact of the released pheromones on the aphids and the ladybugs, we designed an <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator/Modelling" target="_blank">oscillating transcription factor network</a> to regulate their production. This oscillator also communicates between cells, enforcing the oscillating rhythm onto the whole colony.<br/><br />
Summarized, <b>these algorithms allow us to model our system from the cellular metabolism throughout to the environmental impact</b>. Based on our models, we adapted the actual building of the system towards the most effective circuit. Finally, our mathematical predictions will provide significant benefits once we prepare our E. coligy for field tests. <br />
</p><br />
</div><br />
</div><br />
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<div id="header" class="row-fluid"><br />
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<div class="span12 white"><br />
<h3>Ecology</h3><br />
<p align="justify"><b>Two companies specialized in insects were very interested in our project. They invited us to perform experiments at their facilities.</b> These experiments proved that methyl salicylate not only attracts predators but also activates & protects the plant against aphids. They also proved that aphids are driven away by methyl salicylate.<br/><br />
</div><br />
</div><br />
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<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-red">Human Practices</h3><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align="justify">As a result of our <b>unique collaboration between philosophers and scientists within our team, we formulated several new approaches to ethical and philosophical considerations</b>. The best example is the <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Descriptive" target="_blank"> descriptive evaluation</a> in which all team members and supervisors were interviewed about their ethical beliefs. This bottom-up collaboration between students is a new starting point for approaching human practices within the iGEM competition. We also explained why participants should inform the general public using the ideas of philosopher <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/HannahArendt" target="_blank">Hannah Arendt</a> and we examined the responsability of synthetic biologists using the ideas of philosopher <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/HansJonas" target="blank">Hans Jonas</a>. <b>This is not only a first time in the iGEM competition, but has also never been done in the scientific literature!</b><br/><br />
<p align="justify">To make sure that we have <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/EndUser" target="blank">a product that is worth to be launched</a> we asked Biobest & pcfruit why they were so interested in the BanAphids. <b>We also asked farmers whether they would use our BanAphids and the results were definitely positive!</b></p><br />
<br />
</p><br />
</div><br />
</div><br />
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<div id="header" class="row-fluid"><br />
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<p align="justify"><b>We organized a symposium for the general public for which we also invited the other BeNeLux teams to present their project.</b> We hope that one day this will become a yearly tradition in the iGEM competition.<br/><br />
We went to schools to teach them about synthetic biology. For this we made our very own Plexiglas "biobricks" which the students can use to work on excercises and we made a <b>3D-bacterial model</b>, which gives the students an idea of what a bacterium looks like</p><br />
</div><br />
</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T23:23:04Z<p>LaurensDeBacker: </p>
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It is of course very important to stay safe. Here we have gathered information on how we stayed <a href="#general safety">safe in our lab</a>, what the <a href="#europe legislation">government rules</a> and the <a href="#KUL">rules of the KU Leuven</a> are. Finally, we also elaborate on how we could keep our <a href="#bacterial safety">bacterium from spreading</a> if it would eventually be commercialised and released into the <a href="#environmental safety">environment</a>. As well as which specific mechanisms have we implemented to keep our <a href="#auxotrophic">bacteria contained</a>, with for instance, a <a href="#kill switch">'kill switch'</a>. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning certain safety issues.</p><br />
<p align="justify"><br />
Safety forms were <b>approved</b> on September 29, 2013 by the iGEM Safety Committee.<br />
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We have covered the safety aspect of our project in many ways. Within synthetic biology there should be three main risks that need to be covered: exposure of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem.<br />
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<p><i>(Max Gibson)</i></p><br />
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In the past three months, we have added new branches into the pathways of <i>E. coli</i>. Our experiments were performed with <b>non-pathogenic <i>E. coli</i> strains</b> (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms demands extra safety conditions; physical measures have been taken to separate the organism from the surrounding environment and vice versa.<br/><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria (<b>BanAphids</b>) will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are naturally occurring substances. EBF repels aphids and MeS attracts ladybugs, one of the aphid's natural enemies. The low concentrations needed for these substances to be effective is not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with <b>acetylsalicylic acid (aspirin)</b> and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our <b>BanAphids</b> could cause allergic reactions (swelling, redness, ...), but serious reactions only occur in high concentrations and are very rare.<br/><br />
The effect of <b>BanAphids</b> on the environment is not trivial and thorough field experiments must definitely be conducted. The maximum production rate of a single bacterium needs to be determined so that this could be scaled up through a model. This way we would be able to estimate the concentration of EBF and MeS in the surroundings if a whole field would be occupied by <b>BanAphids</b>. We can then determine the necessary parameters in order to minimise ecological disruption according to these calculated estimates of surrounding EBF and MeS concentrations. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present, therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<p align = "justify">Research concerning genetically modified organisms (GMOs) is subjected to strict safety legislation in Belgium. These laws are in place to protect the general public and the environment from the potential risks of GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB) (section Biosafety and Biotechnology of the Scientific Institute of Public Health) which acts as the scientific secretary of the board. They evaluate the risk analysis in the licence request. At the University of Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
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<p><i>(JRC - European Commission)</i></p><br />
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The KU Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for <b>Health, Safety and Environment (HSE)</b> issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the KU Leuven. The safety training consisted of a summary of the important biosafety issues and the composition of the lab. At the beginning of our project, all members of the iGEM team have received an update from the KU Leuven's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br/><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we discussed the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof. Dr. Johan Robben and Dr. Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. They have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. For instance we talked to the company Perseus (Partner in Regulatory and Safety Services) to discuss the possible hurdles we would face and which safety aspects we need to incorporate. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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If our project would be used outside the laboratory, many parameters would need to be determined. As stated earlier, the pheromones produced by the <b>BanAphids</b> are not harmful for our health unless available in high concentrations. So if the <b>BanAphids</b> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
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<p><i>(Norcal)</i></p><br />
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In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested by Moe-Behrens <i>et al.</i>, 2013, containment through engineered auxotrophy so that <b>BanAphids</b> are unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We also believe that providing a bacterium with an advantage eg. antibiotic resistance, provides bacteria with the opportunity to mutate into even stronger bacteria. We therefore propose the use of a tryptophan auxotrophic bacterium.<br/><br />
When <b>tryptophan auxotrophic</b> bacteria will be used in our project, we must provide tryptophan in their medium. This is a good candidate for the biocontainment of <b>BanAphids</b> when the bacteria grow together on a petri dish or another closed environment. For instance, in one of the models we designed where the bacteria are grown in a contained environment, the sticker <a href="https://2012.igem.org/Team:Groningen/Sticker">(Design of Groningen iGEM 2012)</a>. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape. This auxotrophic mechanism, however, does not work when we want to spray the bacteria on the plants and crops.<br/><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition and we find it very important that the work of previous iGEM teams do not go to waste which is why we decided to look into several 'kill switches' developed by previous iGEM teams. We examined the available 'kill switches' and selected the best one for our project.</p><br />
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<p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
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We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesise an essential compound required for their survival, they rapidly die once they escape from the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produces a toxic protein that disrupts the bacterial cell's membrane and kills the cells. A stochastic activation of this toxic component could also be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
This would be easily applicable for the 'sticker' or 'oscillator' model. However, we need a different mechanisms to contain the <b>BanAphids</b> of our 'spray' or 'glucose' model.</p><br />
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The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g. IPTG) is added, and this will activate a cascade mechanism that will kill the cells. This induced lethality could, for instance, be used to clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM, there are already several constructs available in the database, such as the inducible <i>BamHI</i> system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, spontaneous genetic mutations in this lethal <i>BamHI</i> gene is a central cause of safeguard failure. Spontaneous genetic mutations can deactivate lethal gene expression, which cause failure of biocontainment. As these engineered cells propagate in the laboratory, or in large bioreactors, disfunctional genetic safeguards can gradually accumulate in the population. If the accumulation is high enough, then the synthetic organisms might survive in the environment after disposal or accidental release (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Calgary 2012 Team</a>.<br />
Furthermore, as suggested by Balan <i>et al.</i> (2005), the use of a nuclease enzyme suicide system disfavours horizontal gene transfer compared to the above suggested suicide system that targets the cell membrane. If a bacterial cell is lysed, that bacterium might be dead but other bacteria in the environment are not dead (yet) and a suicide system that targets the disruption of the cell membrane does not guarantee the degradation of DNA. The lysed bacterium's DNA is now free and available to be taken up by neighbouring bacteria. For this reason we have chosen a 'kill switch' that activates a nuclease system. We refer here to the <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">kill genes</a> courtesy of the Calgary team of 2012. <br />
</p></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T23:15:13Z<p>LaurensDeBacker: </p>
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It is of course very important to stay safe. Here we have gathered information on how we stayed <a href="#general safety">safe in our lab</a>, what the <a href="#europe legislation">government rules</a> and the <a href="#KUL">rules of the KU Leuven</a> are. Finally, we also elaborate on how we could keep our <a href="#bacterial safety">bacterium from spreading</a> if it would eventually be commercialised and released into the <a href="#environmental safety">environment</a>. As well as which specific mechanisms have we implemented to keep our <a href="#auxotrophic">bacteria contained</a>, with for instance, a <a href="#kill switch">'kill switch'</a>. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning certain safety issues.</p><br />
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Safety forms were <b>approved</b> on September 29, 2013 by the iGEM Safety Committee.<br />
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We have covered the safety aspect of our project in many ways. Within synthetic biology there should be three main risks that need to be covered: exposure of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem.<br />
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In the past three months, we have added new branches into the pathways of <i>E. coli</i>. Our experiments were performed with <b>non-pathogenic <i>E. coli</i> strains</b> (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms demands extra safety conditions; physical measures have been taken to separate the organism from the surrounding environment and vice versa.<br/><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria (<b>BanAphids</b>) will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are naturally occurring substances. EBF repels aphids and MeS attracts ladybugs, one of the aphid's natural enemies. The low concentrations needed for these substances to be effective is not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with <b>acetylsalicylic acid (aspirin)</b> and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our <b>BanAphids</b> could cause allergic reactions (swelling, redness, ...), but serious reactions only occur in high concentrations and are very rare.<br/><br />
The effect of <b>BanAphids</b> on the environment is not trivial and thorough field experiments must definitely be conducted. The maximum production rate of a single bacterium needs to be determined so that this could be scaled up through a model. This way we would be able to estimate the concentration of EBF and MeS in the surroundings if a whole field would be occupied by <b>BanAphids</b>. We can then determine the necessary parameters in order to minimise ecological disruption according to these calculated estimates of surrounding EBF and MeS concentrations. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present, therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<p align = "justify">Research concerning genetically modified organisms (GMOs) is subjected to strict safety legislation in Belgium. These laws are in place to protect the general public and the environment from the potential risks of GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB) (section Biosafety and Biotechnology of the Scientific Institute of Public Health) acts as the scientific secretary of the board. They evaluate the risk analysis in the licence request. At the University of Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
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The KU Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the KU Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KU Leuven's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br/><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we discussed the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof. Dr. Johan Robben and Dr. Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. They have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. For instance we talked to the company Perseus (Partner in Regulatory and Safety Services) to discuss the possible hurdles we would face and which safety aspects we need to incorporate. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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If our project would be used outside the laboratory, many parameters would need to be determined. As stated earlier, the pheromones produced by the <b>BanAphids</b> are not harmful for our health unless available in high concentrations. So if the <b>BanAphids</b> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
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In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested by Moe-Behrens <i>et al.</i>, 2013, containment through engineered auxotrophy so that <b>BanAphids</b> are unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We also believe that providing a bacterium with an advantage eg. antibiotic resistance, provides bacteria with the opportunity to mutate into even stronger bacteria. We therefore propose the use of a tryptophan auxotrophic bacterium.<br/><br />
When <b>tryptophan auxotrophic</b> bacteria will be used in our project, we must provide tryptophan in their medium. This is a good candidate for the biocontainment of <b>BanAphids</b> when the bacteria grow together on a petri dish or another closed environment. For instance, in one of the models we designed where the bacteria are grown in a contained environment, the sticker <a href="https://2012.igem.org/Team:Groningen/Sticker">(Design of Groningen iGEM 2012)</a>. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape. This auxotrophic mechanism, however, does not work when we want to spray the bacteria on the plants and crops.<br/><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition and we find it very important that the work of previous iGEM teams do not go to waste which is why we decided to look into several 'kill switches' developed by previous iGEM teams. We examined the available 'kill switches' and selected the best one for our project.</p><br />
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<p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
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We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesise an essential compound required for their survival, they rapidly die once they escape from the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produces a toxic protein that disrupts the bacterial cell's membrane and kills the cells. A stochastic activation of this toxic component could also be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
This would be easily applicable for the 'sticker' or 'oscillator' model. However, we need a different mechanisms to contain the <b>BanAphids</b> of our 'spray' or 'glucose' model.</p><br />
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<p align="justify"><br />
The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g. IPTG) is added, and this will activate a cascade mechanism that will kill the cells. This induced lethality could, for instance, be used to clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM, there are already several constructs available in the database, such as the inducible <i>BamHI</i> system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, spontaneous genetic mutations in this lethal <i>BamHI</i> gene is a central cause of safeguard failure. Spontaneous genetic mutations can deactivate lethal gene expression, which cause failure of biocontainment. As these engineered cells propagate in the laboratory, or in large bioreactors, disfunctional genetic safeguards can gradually accumulate in the population. If the accumulation is high enough, then the synthetic organisms might survive in the environment after disposal or accidental release (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch">Calgary 2012 Team</a>.<br />
Furthermore, as suggested by (Balan <i>et al.</i>, 2005) the use of a nuclease enzyme suicide system disfavours horizontal gene transfer compared to the above suggest suicide system that targets the cell membrane. If a bacterial cell is lysed, that bacterium might be dead but other bacteria in the environment are not dead (yet) and a suicide system that targets the disruption of the cell membrane does not guarantee the degradation of DNA. The lysed bacterium's DNA is now free and available to be taken up by neighbouring bacteria. For this reason we have chosen a 'kill switch' that activates a nuclease system. We refer here to the <a href="https://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/KillGenes">kill genes</a> courtesy of the Calgary team of 2012. <br />
</p></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/DesignTeam:KU Leuven/Project/Glucosemodel/Design2013-10-04T22:55:44Z<p>LaurensDeBacker: </p>
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We thought of two different methods to carry out our system. One method would be to <b>spray our BanAphids onto the plants</b>. Keeping into account the possible hurdles we mentioned before, BanAphids will produce MeS in response to an external signal that indicates the presence of aphids, in order to reduce the burden on the plant cell’s metabolism and attract predators only when needed. This external signal is <b>honeydew</b>, since aphids produce high amounts of this. Honeydew is a very glucose rich substance, which is the reason why ants ‘farm’ aphids, so they can milk their honeydew.</p><br />
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<p align="justify"> <b>Tet repressor under low glucose promoter</b> </p><br />
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pCaiF is a low glucose promoter, so <b>when aphids are present on the plant and thereby honeydew </b>, TetR will <b>not </b> be transcribed. ptetR, a TetR repressible promoter, will be active in this case so that <I>lacI</I> and <I>aroG*</I> will be transcribed. <I>aroG</I> encodes for the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which will convert erythrose-4-phosphate and phosphoenolpyruvate into 3-deoxy-arabine-heptulosonate-7-phosphate, a precursor of chorismate. <b>We have mutated <I>aroG</I> into <I>aroG*</I> in order to inhibit the negative feedback mechanism of phenylalanine to increase the activity of DAHP synthase so that the chorismate concentration is increased</b>.<br />
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<p align="justify">The following construct will <b> convert the chorismate produced into salicylic acid and then into MeS</b>. The original BioBrick from the 2006 MIT team (<a href="http://parts.igem.org/Part:BBa_J45700">Bba_J45700</a>) contained a <I>lac</I> promoter in front of the <I>pchBA gene</I>. The <I>pchBA</I> gene encodes two enzymes (isochorismate synthase and isochorismate pyruvate/lyase) that convert chorismate into salicylic acid. Since this would interfere with our system (we use LacI), we replaced this promoter with another <i>tetR</i> promoter.</p><br />
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cpram is a constitutive promoter so the EBF synthase gene will be constitutively transcribed and EBF constitutively produced. However, there is a <I>lac</I> operator present and since<b> LacI is transcribed when honeydew is present</b> (see above), <b>EBF synthase transcription is inhibited in the presence of honeydew</b>. In the absence of aphids, EBF is constitutively expressed and aphids are thus repelled. However, as mentioned before, EBF could lose its aphid repellent effect due to habituation.<br />
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<p align="justify">So if certain aphids do happen to escape the EBF repellent signal, the <b>MeS acts as a counter signal</b> and <b>attracts natural predators</b> of the aphid such as ladybugs and green lacewings. <b>Aphids will activate the MeS cycle due to the presence of honeydew</b>.</p><br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/DesignTeam:KU Leuven/Project/Glucosemodel/Design2013-10-04T22:46:44Z<p>LaurensDeBacker: </p>
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We thought of two different methods to carry out our system. One method would be to <b>spray our BanAphids onto the plants</b>. Keeping into account the possible hurdles we mentioned before, BanAphids will produce MeS in response to an external signal that indicates the presence of aphids, in order to reduce the burden on the plant cell’s metabolism and attract predators only when needed. This external signal is <b>honeydew</b>, since aphids produce high amounts of this. Honeydew is a very glucose rich substance, which is the reason why ants ‘farm’ aphids, so they can milk their honeydew.</p><br />
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<p align="justify"> <b>Tet repressor under low glucose promoter</b> </p><br />
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<p align="justify"> <b> AroG*-LacI construct </b> </p><br />
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pCaiF is a low glucose promoter, so <b>when aphids are present on the plant and thereby honeydew </b>, TetR will <b>not </b> be transcribed. ptetR, a TetR repressible promoter, will be active in this case so that <I>lacI</I> and <I>aroG*</I> will be transcribed. <I>aroG</I> encodes for the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which will convert erythrose-4-phosphate and phosphoenolpyruvate into 3-deoxy-arabine-heptulosonate-7-phosphate, a precursor of chorismate. <b>We have mutated <I>aroG</I> into <I>aroG*</I> in order to inhibit the negative feedback mechanism of phenylalanine to increase the activity of DAHP synthase so that the chorismate concentration is increased</b>.<br />
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<p><b>MeS construct to convert salicylic acid into MeS</b></p><br />
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<p align="justify">The following construct will <b> convert the chorismate produced into salicylic acid and then into MeS</b>. The original BioBrick from the 2006 MIT team (<a href="http://parts.igem.org/Part:BBa_J45700">Bba_J45700</a>) contained a <I>lac</I> promoter in front of the <I>pchBA gene</I>. The <I>pchBA</I> gene encodes an enzyme that converts chorismate into salicylic acid. Since this would interfere with our system (we use LacI), we replaced this promoter with another <i>tetR</i> promoter.</p><br />
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cpram is a constitutive promoter so the EBF synthase gene will be constitutively transcribed and EBF constitutively produced. However, there is a <I>lac</I> operator present and since<b> LacI is transcribed when honeydew is present</b> (see above), <b>EBF synthase transcription is inhibited in the presence of honeydew </b>. In the absence of aphids, EBF is constitutively expressed and aphids are thus repelled. However, as mentioned before, EBF could lose its aphid repellent effect due to habituation.<br />
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<p><b>EBF construct</b></p><br />
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<p align="justify">So if certain aphids do happen to escape the EBF repellent signal, the <b> MeS acts as a counter signal </b> and <b>attracts natural predators </b> of the aphid such as ladybugs and green lacewings. <b>Aphids will activate the MeS cycle due to the presence of honeydew</b>.</p><br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Project/HoneydewSystemTeam:KU Leuven/Project/HoneydewSystem2013-10-04T22:13:28Z<p>LaurensDeBacker: </p>
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We aim to achieve a sustainable way to reduce the damage caused by aphid pests, and offer an effective alternative for insecticides. Our modified <I>E. coli</I>, the <b>BanAphid</b> (meaning ‘to ban aphids’ as well as with ‘benefits’), would imitate insecticides by using the aphid’s own alarm pheromone, <b>E-β-farnesene</b>, (EBF) to <b>repel them off the plant</b>. On top of that we want to <b>attract aphid predators</b> such as the ladybug by using <b>methyl salicylate</b> (MeS), a phytohormone. This way we make sure the aphids are thoroughly removed from the plant.<br/><br />
We have established what might be possible hurdles in introducing this system in the agricultural industry. First we have to make sure that the <b>plant cell’s metabolism is not over burdened</b>. Besides that we have to take into account that aphids might <b>habituate</b> to constitutive expression of EBF (De Vos <I>et al.</I>, 2010, Kunert <I>et al.</I>, 2010). Finally, we do not want to attract the aphid’s natural predators when they are not needed. <br/></p><br />
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<p align="justify">The first glucose model we present is spraying our BanAphids onto the plants. Aphids activate the methyl salicylate cycle due to the presence of honeydew. If certain aphids do happen to escape the EBF repellent signal, this methyl salicylate acts as a counter signal and attracts natural predators of the aphids such as ladybugs and green lacewings. </p><br />
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<p align="justify">EBF serves as the most universal aphid alarm pheromone. It is released from the cornicles of the aphids to warn others against upcoming danger, such as the natural predators of aphids. Because of the fact that EBF is highly susceptible to oxidation, we want to make our BanAphids produce EBF regularly.</p><br />
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<p align="justify">Methyl salicylate is a pheromone released by plants when they are attacked by aphids. It activates plant defence systems, as well as attract predators of the aphids, such as the ladybug or the green lacewing. In the lab we have focussed on increasing the production of methyl salicylate of an existing brick, by increasing the production of its precursor, chorismate.</p><br />
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<p align="justify">We ran the Flux Balancing Analysis using the COBRA Toolbox for MATLAB. With this analysis we checked whether the growth rate of our cells will be affected when we introduce our methyl salicylate system.</p><br />
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<p align="justify">We have modelled the production system of methyl salicylate by using the transcription, translation and protein degradation rates in order to calculate the mRNA and protein fluxes. We also brought the kinetics of methyl salicylate synthesis into account.</p><br />
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<p align="justify">We did a qPCR to check whether our genes of interest are properly transcribed. Also, the amount of mRNA we can measure with qPCR could serve as input data for our methyl salicylate model.</p><br />
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<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
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<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
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<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
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<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
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<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
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<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
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<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
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<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
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<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a popular Belgian scientific magazine, publishing monthly issues. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
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<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas, Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from pcfruit. Pcfruit is a station for research and advisory services of fruit growing, situated on three locations in Limburg ,a province of Belgium. They deal with a range of research activities concerning the most productive varieties of cherries, strawberries, apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed, and got to taste some fresh apple juice while we presented our project. We were glad to receive some thoughtful remarks and advice from people with good expertise, and we're looking forward to a possible collaboration for some experiments in the near future.<br />
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<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
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<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
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<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers what they thought of our project, since they are our main target audience. The results can be found <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/EndUser">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
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<h3>Modelling</h3> </a><br />
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<p align="justify">This summer, three dedicated male engineers spent a whole summer modelling our systems. <br/>However, without the help of a girl, Tina, this would have been a rather ''nerdy'' journal. Luckily, Tina was always there when the modelling team sought a balance between awful jokes and work efficiency. Nevertheless, without Sander's work tempo, Bert’s criticism and Tomas' factotum mind, the modelling would not have been as thorough as now.<br/><br />
In this journal, you can read how they started modelling from scratch up to being modelling specialists, using Matlab, Symbiology, COBRA and even qPCR.</p><br />
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<h3>Ecology</h3> </a><br />
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<p align="justify">We worked together with two research groups, Biobest and pcfruit. Both offer a whole series of parasitoids and predators against aphids for the purpose of biological pest control and investigate the relationship between plant and aphid in search of a method to protect plants from this pest. Here, you can read more about the experiments Aurélie and Flore (Biobest) and Tina and Tomas (pcfruit) performed to test our model on aphids and ladybugs.</p><br />
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<h3>E-β-Farnesene</h3></a><br />
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<p align="justify">Here we present the work on the <a href="Http://2013.igem.org/Team:KU Leuven/Project/Glucosemodel/EBF">EBF-biobrick</a>, a part of the glucose model, week by week. The contributors are Lukas and Su, which formed a very tight team that already started with lab work even during the exams in June. Lukas’ multitasking skills and Su’s precise work, combined with their cute smiles, made sure everyone regretted not being part of this team. <br/> You can read all about the extensive work the EBF guys have done in this part.</p><br />
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<h3>Feed forward loop</h3> </a><br />
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<p align="justify">Long team discussions and Bert’s persistence resulted this summer in a ‘proof of concept’ wetlab version of Bert Mortier’s oscillator, called the ‘feed forward loop’. <br/><br />
Pieter and Sylvie were immediately up to form a subteam and started working in order to create a part of the <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">oscillator</a>.<br/><br />
In this part, you can read all about their big UP in the beginning, going to a down after a while and the eventual steady state with troubleshooting in the end.<br/><br />
Nevertheless, this feed forward loop is only the beginning to create Bert’s oscillator.</p><br />
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<p align="justify">The Methyl Salicylate team or ‘MeS boys’, in normal life called Robbert and Laurens, worked on the improvement of the methyl salicylate brick round the clock almost whole summer. The journal can be found here. More information on the MeS biobrick can be found on <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS"> this page</a>.</p><br />
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<p align="justify">The modelling team also performed a qPCR, a new and appeasing way to characterise and to find data for our <a href="https://2013.igem.org/Team:KU_Leuven/Project/qPCR">computer model</a>. In this part, you can read how to properly manage with troubles and how to search for promising directions.</p><br />
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<h3>Modelling</h3> </a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">This summer, three dedicated male engineers spent a whole summer modelling our systems. <br/>However, without the help of a girl, Tina, this would have been a rather ''nerdy'' journal. Luckily, Tina was always there when the modelling team sought a balance between awful jokes and work efficiency. Nevertheless, without Sanders work tempo, Bert’s criticism and Tomas' factotum mind, the modelling would not have been as thorough as now.<br/><br />
In this journal, you can read how they started modelling from scratch up to being modelling specialists, using Matlab, Symbiology, COBRA and even qPCR.</p><br />
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<div class="span10 icon-text "><br />
<h3>Ecology</h3> </a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">We worked together with two research groups, Biobest and pcfruit. Both offer a whole series of parasitoids and predators against aphids for the purpose of biological pest control and investigate the relationship between plant and aphid in search of a method to protect plants from this pest. Here, you can read more about the experiments Aurélie and Flore (Biobest) and Tina and Tomas (pcfruit) performed to test our model on aphids and ladybugs.</p><br />
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<i class="livicon activeicon" data-name="bell" data-onparent="true" data-color="white"></i><br />
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<br />
<div class="span10 icon-text "><br />
<h3>E-β-Farnesene</h3></a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">Here we present the work on the <a href="Http://2013.igem.org/Team:KU Leuven/Project/Glucosemodel/EBF">EBF-biobrick</a>, a part of the glucose model, week by week. The contributors are Lukas and Su, which formed a very tight team that already started with lab work even during the exams in June. Lukas’ multitasking skills and Su’s precise work, combined with their cute smiles, made sure everyone regretted not being part of this team. <br/> You can read all about the extensive work the EBF guys have done in this part.</p><br />
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<br />
<div class="span10 icon-text "><br />
<h3>Feed forward loop</h3> </a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">Long team discussions and Bert’s persistence resulted this summer in a ‘proof of concept’ wetlab version of Bert Mortier’s oscillator, called the ‘feed forward loop’. <br/><br />
Pieter and Sylvie were immediately up to form a subteam and started working in order to create a part of the <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">oscillator</a>.<br/><br />
In this part, you can read all about their big UP in the beginning, going to a down after a while and the eventual steady state with troubleshooting in the end.<br/><br />
Nevertheless, this feed forward loop is only the beginning to create Bert’s oscillator.</p><br />
</div><br />
</div><br />
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<h3>Methyl Salicylate</h3> </a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">The Methyl Salicylate team or ‘MeS boys’, in normal life called Robbert and Laurens, worked on the improvement of the methyl salicylate brick round the clock almost whole summer. The journal can be found here. More information on the MeS biobrick can be found on <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS"> this page</a>.</p><br />
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<br />
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<h3>qPCR</h3> </a><br />
<div class="row-fluid"><br />
<div class="span12"><br />
<p align="justify">The modelling team also performed a qPCR, a new and appeasing way to characterise and to find data for our <a href="https://2013.igem.org/Team:KU_Leuven/Project/qPCR">computer model</a>. In this part, you can read how to properly manage with troubles and how to search for promising directions.</p><br />
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<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
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<p>Follow our activities week-by-week.</p><br />
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<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
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<p>Be careful!</p><br />
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<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
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<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
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</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
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<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
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</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
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<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
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<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
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<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine which publishes five editions each year. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
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<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas, Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from pcfruit. Pcfruit is a station for research and advisory services of fruit growing, situated on three locations in Limburg ,a province of Belgium. They deal with a range of research activities concerning the most productive varieties of cherries, strawberries, apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed, and got to taste some fresh apple juice while we presented our project. We were glad to receive some thoughtful remarks and advice from people with good expertise, and we're looking forward to a possible collaboration for some experiments in the near future.<br />
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<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
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<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
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<h3>Doing some inquiries.</h3><br />
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<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers what they thought of our project, since they are our main target audience. The results can be found <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/EndUser">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
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<p align="justify">This summer, three dedicated male engineers spent a whole summer modelling our systems. <br/>However, without the help of a girl, Tina, this would have been a rather ''nerdy'' journal. Luckily, Tina was always there when the modelling team sought a balance between awful jokes and work efficiency. Nevertheless, without Sanders work tempo, Bert’s criticism and Tomas' factotum mind, the modelling would not have been as thorough as now.<br/><br />
In this journal, you can read how they started modelling from scratch up to being modelling specialists, using Matlab, Symbiology, COBRA and even qPCR.</p><br />
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<p align="justify">We worked together with two research groups, Biobest and pcfruit. Both offer a whole series of parasitoids and predators against aphids for the purpose of biological control of pests and investigate the relationship between plant and aphid in search of a method to protect plants from this pest. Here, you can read more about the experiments Aurélie and Flore (Biobest) and Tina and Tomas (pcfruit) performed to test our model on aphids and ladybugs.</p><br />
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<p align="justify">Here we present the work on the <a href="Http://2013.igem.org/Team:KU Leuven/Project/Glucosemodel/EBF">EBF-biobrick</a>, a part of the glucose model, week by week. The contributors are Lukas and Su, which formed a very tight team that already started with lab work even during the exams in June. Lukas’ multitasking skills and Su’s precise work, combined with their cute smiles, made sure everyone regretted not being part of this team. <br/> You can read all about the extensive work the EBF guys have done in this part.</p><br />
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<p align="justify">Long team discussions and Bert’s persistence resulted this summer in a ‘proof of concept’ wetlab version of Bert Mortier’s oscillator, called the ‘feed forward loop’. <br/><br />
Pieter and Sylvie were immediately up to form a subteam and started working in order to create a part of the <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">oscillator</a>.<br/><br />
In this part, you can read all about their big UP in the beginning, going to a down after a while and the eventual steady state with troubleshooting in the end.<br/><br />
Nevertheless, this feed forward loop is only the beginning to create Bert’s oscillator.</p><br />
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<p align="justify">The Methyl Salicylate team or ‘MeS boys’, in normal life called Robbert and Laurens, worked on the improvement of the methyl salicylate brick round the clock almost whole summer. The journal can be found here. More information on the MeS biobrick can be found on <a href="https://2013.igem.org/Team:KU_Leuven/Project/Glucosemodel/MeS"> this page</a>.</p><br />
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<p align="justify">The modelling team also performed a qPCR, a new and appeasing way to characterise and to find data for our <a href="https://2013.igem.org/Team:KU_Leuven/Project/qPCR">computer model</a>. In this part, you can read how to properly manage with troubles and how to search for promising directions.</p><br />
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<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
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<p>No magic involved - unfortunately</p><br />
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<p>Be careful!</p><br />
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<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
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<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
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<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
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<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
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<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
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<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
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<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
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<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
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<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine which publishes five editions each year. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
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<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas, Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a station for research and advisory services of fruit growing, situated on three locations in Limburg ,a province of Belgium. They deal with a range of research activities concerning the most productive varieties of cherries, strawberries, apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed, and got to taste some fresh apple juice while we presented our project. We were glad to receive some thoughtful remarks and advice from people with good expertise, and we're looking forward to a possible collaboration for some experiments in the near future.<br />
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<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
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<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
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<h3>Doing some inquiries.</h3><br />
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<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers what they thought of our project, since they are our main target audience. The results can be found <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/EndUser">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
</div><br />
</div><br />
<br />
<!--LOGOS--><br />
<br />
<br />
<div class="row-fluid"><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-journals"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow1"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
</div><br />
</div><br />
</div> <br />
<br />
<br />
<!-- Team Journal --> <br />
</div><br />
<br />
<body><br />
<div id="container"><br />
<div class="container"><br />
<div class="row-fluid"><br />
<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine which publishes five editions each year. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas, Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a station for research and advisory services of fruit growing, situated on three locations in Limburg ,a province of Belgium. They deal with a range of research activities concerning the most productive varieties of cherries, strawberries, apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed, and got to taste some fresh apple juice while we presented our project. We were glad to receive some thoughtful remarks and advice from people with good expertise, and we're looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers what they thought of our project, since they are our main target audience. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<script><br />
$('.cbp_tmlabel').click(function(){<br />
console.log($(this).children("div").toggle('slow'));<br />
});<br />
</script><br />
</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/NotebookTeam:KU Leuven/Notebook2013-10-04T20:35:41Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
{{:Team:KU Leuven/Template:Style}}<br />
{{:Team:KU Leuven/Template:Menu}}<br />
<html><br />
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<div id="container"><br />
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<!-- TITLE --><br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Notebook</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
</div><br />
</div><br />
<br />
<!--LOGOS--><br />
<br />
<br />
<div class="row-fluid"><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-journals"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow1"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
</div><br />
</div><br />
</div> <br />
<br />
<br />
<!-- Team Journal --> <br />
</div><br />
<br />
<body><br />
<div id="container"><br />
<div class="container"><br />
<div class="row-fluid"><br />
<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine which publishes five editions each year. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas,Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a research station for research and advisory services of fruit growing situated on three locations in Limburg ( a region in Belgium). They offer a range of research activities concerning the most productive varieties of cherries, strawberries apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed and got to taste the fresh apple juice, while we could present our project. We were glad to receive some thoughtful remarks and advice from people with good expertise and are looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers, our main target audience what they thought of our project. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<script><br />
$('.cbp_tmlabel').click(function(){<br />
console.log($(this).children("div").toggle('slow'));<br />
});<br />
</script><br />
</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/NotebookTeam:KU Leuven/Notebook2013-10-04T20:32:54Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
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{{:Team:KU Leuven/Template:Menu}}<br />
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<br />
<!-- TITLE --><br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Notebook</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
</div><br />
</div><br />
<br />
<!--LOGOS--><br />
<br />
<br />
<div class="row-fluid"><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-journals"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow1"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
</div><br />
</div><br />
</div> <br />
<br />
<br />
<!-- Team Journal --> <br />
</div><br />
<br />
<body><br />
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<div class="container"><br />
<div class="row-fluid"><br />
<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different, and so we learned from each other. They already had a nice overview of the literature, while we already did a lot of work for sponsoring, modelling and human practices. We made a common press article, and hopefully we'll get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine and has five editions. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas,Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a research station for research and advisory services of fruit growing situated on three locations in Limburg ( a region in Belgium). They offer a range of research activities concerning the most productive varieties of cherries, strawberries apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed and got to taste the fresh apple juice, while we could present our project. We were glad to receive some thoughtful remarks and advice from people with good expertise and are looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers, our main target audience what they thought of our project. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<script><br />
$('.cbp_tmlabel').click(function(){<br />
console.log($(this).children("div").toggle('slow'));<br />
});<br />
</script><br />
</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/NotebookTeam:KU Leuven/Notebook2013-10-04T20:29:42Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
{{:Team:KU Leuven/Template:Style}}<br />
{{:Team:KU Leuven/Template:Menu}}<br />
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<br />
<!-- TITLE --><br />
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<h3 class="bg-yellow">Notebook</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
</div><br />
</div><br />
<br />
<!--LOGOS--><br />
<br />
<br />
<div class="row-fluid"><br />
<br />
<div class="span4 icon white"><br />
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<div class="visible-desktop span3 bg-journals"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow1"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
</div><br />
</div><br />
</div> <br />
<br />
<br />
<!-- Team Journal --> <br />
</div><br />
<br />
<body><br />
<div id="container"><br />
<div class="container"><br />
<div class="row-fluid"><br />
<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven, and after a few Belgian bears, they were willing to give us lots of tips & tricks to get through iGEM this summer. We noticed that the Belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different and so we learned from each other. They had already a nice overview of the literature and we already did a lot of work for sponsoring, modelling and human practices. We made a common press article and hopefully we get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine and has five editions. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas,Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a research station for research and advisory services of fruit growing situated on three locations in Limburg ( a region in Belgium). They offer a range of research activities concerning the most productive varieties of cherries, strawberries apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed and got to taste the fresh apple juice, while we could present our project. We were glad to receive some thoughtful remarks and advice from people with good expertise and are looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers, our main target audience what they thought of our project. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<script><br />
$('.cbp_tmlabel').click(function(){<br />
console.log($(this).children("div").toggle('slow'));<br />
});<br />
</script><br />
</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/NotebookTeam:KU Leuven/Notebook2013-10-04T20:27:32Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
{{:Team:KU Leuven/Template:Style}}<br />
{{:Team:KU Leuven/Template:Menu}}<br />
<html><br />
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<br />
<!-- TITLE --><br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Notebook</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
</div><br />
</div><br />
<br />
<!--LOGOS--><br />
<br />
<br />
<div class="row-fluid"><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-journals"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow1"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
</div><br />
</div><br />
</div> <br />
<br />
<br />
<!-- Team Journal --> <br />
</div><br />
<br />
<body><br />
<div id="container"><br />
<div class="container"><br />
<div class="row-fluid"><br />
<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason, Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise, but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there! Besides an interesting discussion we also got a guided tour of the facilities. Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven and, after a few belgian bears, were willing to give us lots of tips & tricks to get through iGEM summer. We noticed that the belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different and so we learned from each other. They had already a nice overview of the literature and we already did a lot of work for sponsoring, modelling and human practices. We made a common press article and hopefully we get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine and has five editions. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas,Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a research station for research and advisory services of fruit growing situated on three locations in Limburg ( a region in Belgium). They offer a range of research activities concerning the most productive varieties of cherries, strawberries apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed and got to taste the fresh apple juice, while we could present our project. We were glad to receive some thoughtful remarks and advice from people with good expertise and are looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers, our main target audience what they thought of our project. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
<br />
<br />
</div><br />
</div><br />
</body><br />
<script><br />
$('.cbp_tmlabel').click(function(){<br />
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</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/NotebookTeam:KU Leuven/Notebook2013-10-04T20:22:52Z<p>LaurensDeBacker: </p>
<hr />
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<p align = "justify">In our notebook we collected the journals of our different subteams for you to see what kind of work we did. If you're interested to repeat any of this or use something, check out the protocols page. Working with bioagentia does pose some threats, it is important to stay safe so take a look at our safety page. At the bottom of this page there is some more information on the fun stuff we did this summer.</p><br />
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<a href="https://2013.igem.org/Team:KU_Leuven/Journal" title="Team:KU_leuven"><br />
<i class="livicon activeicon" data-name="calendar" data-color="white"></i></div><br />
<div class="span7 icon-text"><br />
<h3>Journals</h3><br />
</a><br />
<p>Follow our activities week-by-week.</p><br />
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<a href="https://2013.igem.org/Team:KU_Leuven/Protocols"><br />
<i class="livicon activeicon" data-name="magic" data-color="white"></i></div><br />
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<h3>Protocols</h3> </a><br />
<p>No magic involved - unfortunately</p><br />
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<div class="span4 icon white"><br />
<div class="row-fluid"><br />
<div class="visible-desktop span3 bg-yellow"><br />
<a href="https://2013.igem.org/Team:KU_Leuven/Safety"><br />
<i class="livicon activeicon" data-name="biohazard" data-color="white"></i><br />
</div><br />
<div class="span7 icon-text"><br />
<h3>Safety</h3><br />
</a><br />
<p>Be careful!</p><br />
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<body><br />
<div id="container"><br />
<div class="container"><br />
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<div class="span12 timeline"><br />
<ul class="cbp_tmtimeline"><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="caret-right" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Kicking off!</h3><br />
<div class="content"><p align="justify">Today, everyone finally finished their exams, and we decided to get to work immediately so we held a kick-off meeting, so we had our noses all in the same direction.</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>09/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to Biobest</h3><br />
<div class="content"><p align="justify">It is always necessary to get feedback from important players in the field, in order to get relevant feedback on your project. With that thought in mind we contacted <a href="http://www.biobest.be/home/3"> Biobest</a>.<br/><br />
Biobest is a Belgian company which exports bumblebees to over 50 countries worldwide. This makes them the leading authority in biological pollination. The presence of the bumblebees makes it necessary to avoid the use of chemical insecticides. For this reason. Biobest produces and commercializes more than 30 kinds of beneficial insects and mites, which are used for biological crop protection. This made them the ideal company to contact for advise and expertise.<br/><br />
After signing in at the reception we presented our project to prof. Felix Wäckers, their head of R&D. Not only did he give some excellent advise but with his help it will be possible to perform experiments with both ladybugs, aphids and perhaps even other insects.<br/><br />
But our visit didn’t end there, besides an interesting discussion we also got a guided tour of the facilities! Needless to say we are very excited to get to work there!</p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>14/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-down" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We had a visit</h3><br />
<div class="content"><p align="justify">Members of this and previous years' Calgary iGEM team came to visit Leuven and, after a few belgian bears, were willing to give us lots of tips & tricks to get through iGEM summer. We noticed that the belgian beers were a bit harder than they could handle ;-)<br/><br />
<img src="https://static.igem.org/mediawiki/2013/e/e3/Calgaryvisit.jpg" alt="meeting Calgary"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>24/07</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="plane-up" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Going on a visit</h3><br />
<div class="content"><p align="justify">Collaboration with other teams is important, so we went to Ghent to visit our <a href="https://2013.igem.org/Team:UGent">colleagues of the University of Ghent</a>. First, we went to the Faculty of Bioscience engineering, but everything was closed cause of the Gentse Feesten. We met four friendly students of the team and took some time to know each other. We talked about our projects and exchanged some ideas. Our view on the project is totally different and so we learned from each other. They had already a nice overview of the literature and we already did a lot of work for sponsoring, modelling and human practices. We made a common press article and hopefully we get synthetic biology and our projects in the picture here in Belgium. We’ll see each other soon again at Marktrock in Leuven.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/2/29/Meeting_Ghent.JPG" alt="meeting Ghent"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>01/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="truck" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>A big delivery arrived</h3><br />
<div class="content"><p align="justify">Some new reading material has arrived, thanks to our sympathizer Eos! Eos is a Belgian popular scientific magazine and has five editions. They featured our story in one of their publications.<br/><br />
<img src="https://static.igem.org/mediawiki/2013/3/37/Eoses.jpg" alt="Eos delivery"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>08/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="bug" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Visit to pcfruit</h3><br />
<div class="content"><p align="justify">Today Lukas,Sylvie, Sander and Tina had a meeting with Tim Beliën and Dany Bylemans from PCFruit. PCFruit is a research station for research and advisory services of fruit growing situated on three locations in Limburg ( a region in Belgium). They offer a range of research activities concerning the most productive varieties of cherries, strawberries apples or pears, biological fruit growing, the realisation of high quality fruit with a minimum of spraying, the art of attracting birds of prey, predatory mites and bugs and so on. We were warmly welcomed and got to taste the fresh apple juice, while we could present our project. We were glad to receive some thoughtful remarks and advice from people with good expertise and are looking forward to a possible collaboration for some experiments in the near future.<br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>13/08</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="brush" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>We are being artistic!</h3><br />
<div class="content"><p align="justify">We're not only working in the lab or on our computers... Can you guess what we're doing?<br/><br />
<img src="https://static.igem.org/mediawiki/2013/d/d8/Whatisit.jpg" alt="Us being artistic"/></p></div><br />
</div><br />
</li><br />
<br />
<li><br />
<time class="cbp_tmtime"><span></span> <span>02/09</span></time><br />
<div class="cbp_tmicon cbp_tmicon-phone"><i class="livicon activeicon" data-name="pen" data-size="23" data-onparent="true" data-color="white"></i></div><br />
<div class="cbp_tmlabel"><br />
<h3>Doing some inquiries.</h3><br />
<div class="content"><br />
<p align="justify">Today was the yearly farmers' fair in Leuven, an opportunity we couldn't pass. We went out and asked the farmers, our main target audience what they thought of our project. The results can be found <a href="#">here</a>.</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/5/5c/Farmer%27s_inquiry.JPG" width="350"></img><p align="justify"> Aurelie and Tomas talking with a farmer</p><br/><br />
<img src="https://static.igem.org/mediawiki/2013/0/00/Sweet_animals.JPG" alt="sweet sheep" width="200"><p align="justify">Some lovely soft sheep at the fair</p><br />
<br />
<br />
</div><br />
</div><br />
</li><br />
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</div><br />
</body><br />
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$('.cbp_tmlabel').click(function(){<br />
console.log($(this).children("div").toggle('slow'));<br />
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</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/Human_OutreachTeam:KU Leuven/Human Outreach2013-10-04T20:09:00Z<p>LaurensDeBacker: </p>
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<p align = "justify">Synthetic biology isn’t well-known to the general public and we would like to increase awareness concerning its applications and safety. We decided to work on different areas. We organized a symposium, gave workshops in secondary schools, and went to farmers to talk about the project and about synthetic biology in general. We also sent press texts to publicise both synthetic biology and iGEM.</p><br />
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<i class="livicon activeicon" data-name="briefcase" data-color="white"></i></div><br />
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<h3>Education</h3><br />
</a><br />
<p>Ensuring the future of science.</p><br />
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<a href="https://2013.igem.org/Team:KU_Leuven/Human_Outreach/Seminars"><br />
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<h3>Seminars</h3> </a><br />
<p>Lectures for the general public.</p><br />
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<div class="span4 icon white"><br />
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<a href="https://2013.igem.org/Team:KU_Leuven/Human_Outreach/Press"><br />
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<h3>Press</h3><br />
</a><br />
<p>We were in the news!</p><br />
</div><br />
</div><br />
</div> <br />
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</div><br />
<br />
</div><br />
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</body><br />
</html></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/PartsTeam:KU Leuven/Parts2013-10-04T19:58:12Z<p>LaurensDeBacker: </p>
<hr />
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<!-- INTRO --><br />
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<div class="row-fluid"><br />
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<p align = "justify">Here you will find the BioBricks we made this summer in the wetlab. Our three favorite parts are marked with a heart:<br/><br />
<ol><br />
<li> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002">BBa_K1060002</a> was a milestone in our project work. We succeeded to clone the beta farnesene synthase of <i>Artemisia annua</i> in <i>E. coli</i>. To apply to the iGEM standards, we removed an <i>EcoRI</i> site in the gene (AY835398.1).</li><br /><br />
<br />
<li> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009">BBa_K1060009</a> is a construct that constitutively expresses beta farnesene synthase. This synthase will convert farnesyl pyrophosphate into the aphid alarm hormone, (E)-β-farnesene.</li><br /><br />
<br />
<li> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011">BBa_K1060011</a> is similar to BBa_K1060009. However, in this biobrick we added a <i>lac</i> operator in front of the beta farnesene synthase. This makes it possible to switch of (E)- β-farnesene production by using biosensors expressing LacI.</li><br /><br />
</ol><br />
</html><groupparts>iGEM013 KU_Leuven</groupparts><html><br />
</p><br />
</div><br />
</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/PartsTeam:KU Leuven/Parts2013-10-04T19:55:59Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
{{:Team:KU Leuven/Template:Style}}<br />
{{:Team:KU Leuven/Template:Menu}}<br />
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<h3 class="bg-green">Parts</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">Here you will find the BioBricks we made this summer in the wetlab. Our three favorite parts are marked with a heart:<br/><br />
<ol><br />
<li> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002">BBa_K1060002</a> was a milestone in our project work. We succeeded to clone the beta farnesene synthase of <i>Artemisia annua</i> in <i>E. coli</i>. To apply to the iGEM standards, we removed an <i>EcoRI</i> site in the gene (AY835398.1).</li><br /><br />
<br />
<li> BBa_K1060009 is a construct that constitutively expresses beta farnesene synthase. This synthase will convert farnesyl pyrophosphate into the aphid alarm hormone, (E)-β-farnesene.</li><br /><br />
<br />
<li> BBa_K1060011 is similar to BBa_K1060009. However, in this biobrick we added a <i>lac</i> operator in front of the beta farnesene synthase. This makes it possible to switch of (E)- β-farnesene production by using biosensors expressing LacI.</li><br /><br />
</ol><br />
</html><groupparts>iGEM013 KU_Leuven</groupparts><html><br />
</p><br />
</div><br />
</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/PartsTeam:KU Leuven/Parts2013-10-04T19:51:45Z<p>LaurensDeBacker: </p>
<hr />
<div>{{:Team:KU Leuven/Template:Header}}<br />
{{:Team:KU Leuven/Template:Style}}<br />
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<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-green">Parts</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- INTRO --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align = "justify">Here you will find the BioBricks we made this summer in the wetlab. Our three favorite parts are marked with a heart:<br/><br />
<ol><br />
<li> BBa_K1060002 was a milestone in our project work. We succeeded to clone the beta farnesene synthase of <i>Artemisia annua</i> in <i>E. coli</i>. To apply to the iGEM standards, we removed an <i>EcoRI</i> site in the gene (AY835398.1).</li><br /><br />
<br />
<li> BBa_K1060009 is a construct that constitutively expresses beta farnesene synthase. This synthase will convert farnesyl pyrophosphate into the aphid alarm hormone, (E)-β-farnesene.</li><br /><br />
<br />
<li> BBa_K1060011 is similar to BBa_K1060009. However, in this biobrick we added a <i>lac</i> operator in front of the beta farnesene synthase. This makes it possible to switch of (E)- β-farnesene production by using biosensors expressing LacI.</li><br /><br />
</ol><br />
</html><groupparts>iGEM013 KU_Leuven</groupparts><html><br />
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</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/PartsTeam:KU Leuven/Parts2013-10-04T19:50:46Z<p>LaurensDeBacker: </p>
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<p align = "justify">Here you will find the BioBricks we made this summer in the wetlab. Our three favorite parts are marked with a heart:<br/><br />
<ol><br />
<li> BBa_K1060002 was a milestone in our project work. We succeeded to clone the beta farnesene synthase of <i>Artemisia annua</i> in <i>E. coli</i>. To apply to the iGEM standards, we removed an <i>EcoRI</i> site in the gene (AY835398.1).</li><br /><br />
<br />
<li> BBa_K1060009 is a construct that constitutively expresses beta farnesene synthase. This synthase will convert farnesyl pyrophosphate into the aphid alarm hormone, (E)-β-farnesene.</li><br /><br />
<br />
<li> BBa_K1060011 is similar to BBa_K1060009. However, in this biobrick we added a <lac> operator in front of the beta farnesene synthase. This makes it possible to switch of (E)- β-farnesene production by using biosensors expressing LacI.</li><br /><br />
</ol><br />
</html><groupparts>iGEM013 KU_Leuven</groupparts><html><br />
</p><br />
</div><br />
</div></div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T19:35:01Z<p>LaurensDeBacker: </p>
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<p align="justify"><br />
It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p><br />
<p align="justify"><br />
Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.<br />
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We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/><br />
We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.<br />
</div><br />
<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/6/66/Laurens_labo.png" alt="Laurens Labo"><br />
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<img src="https://static.igem.org/mediawiki/2013/f/ff/Ladybug_full.png" alt="some_text"><br />
<p><i>(Max Gibson)</i></p><br />
</div><br />
<br />
<div class="span9"><br />
<p align="justify"><br />
In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms requires extra safety conditions; physical measures are taken to separate the organism from the surrounding environment and vice versa.<br><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br><br />
The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of one bacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<h3>Legislation in Belgium & Europe</h3><br />
<div class="row-fluid"><br />
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<p align = "justify"><br />
In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3e/European_figure.jpg" alt="some_text"><br />
<p><i>(JRC - European Commission)</i></p><br />
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</div><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>University of Leuven rules</h3><br />
<p align="justify"><br />
The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KUL's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/e/ec/Ladybug_tree.png"><br />
<p><i>(Norcal)</i></p><br />
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<h3 class="bg-yellow">Bacterial safety</h3><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
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<p align = "justify"><br />
In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (Moe-Behrens <i>et al.</i>, 2013), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br><br />
When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium. Therefore, an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would choose for this method, we have to examine all the available kill switches and select the best one.</p><br />
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<h3>Auxotrophic mechanism</h3><br />
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<p align="justify"><br />
We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/><br />
However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png" alt="some_text"/><br/><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f2/Spray_antiaphid.png" alt="some_text"/><br />
</div><br />
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<div class="span9"><br />
<h3>Kill Switch</h3><br />
<p align="justify"><br />
The basic mechanism of a kill switch is that the engineered organisms survive until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression, which may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which is a small piece of mRNA that binds ligands and modifies translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/Hum</div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T19:23:41Z<p>LaurensDeBacker: </p>
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<h3 class="bg-yellow">Safety</h3><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align="justify"><br />
It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p><br />
<p align="justify"><br />
Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">General Safety</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- TEKST --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<div class="row-fluid"><br />
<div class="span9"><br />
<p align = "justify"><br />
We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/><br />
We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.<br />
</div><br />
<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/6/66/Laurens_labo.png" alt="Laurens Labo"><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<a id="safety compounds"></a><br />
<div class="row-fluid"><br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/f/ff/Ladybug_full.png" alt="some_text"><br />
<p><i>(Max Gibson)</i></p><br />
</div><br />
<br />
<div class="span9"><br />
<p align="justify"><br />
In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms requires extra safety conditions; physical measures are taken to separate the organism from the surrounding environment and vice versa.<br><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br><br />
The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of one bacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">General safety</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- TEKST --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>Legislation in Belgium & Europe</h3><br />
<div class="row-fluid"><br />
<div class="span9"<br />
<p align = "justify"><br />
In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3e/European_figure.jpg" alt="some_text"><br />
<p><i>(JRC - European Commission)</i></p><br />
</div><br />
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</div><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>University of Leuven rules</h3><br />
<p align="justify"><br />
The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KUL's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
</div><br />
</div><br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
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If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
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<img src="https://static.igem.org/mediawiki/2013/e/ec/Ladybug_tree.png"><br />
<p><i>(Norcal)</i></p><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
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<div class="span6"><br />
<p align = "justify"><br />
In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (Moe-Behrens <i>et al.</i>, 2013), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br><br />
When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium therefore an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would chose for this method, we have to examine all the available kill switches and select the best one.</p><br />
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<h3>Auxotrophic mechanism</h3><br />
<div class="row-fluid"><br />
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<div class="span9"><br />
<p align="justify"><br />
We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/><br />
However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png" alt="some_text"/><br/><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f2/Spray_antiaphid.png" alt="some_text"/><br />
</div><br />
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<div class="span9"><br />
<h3>Kill Switch</h3><br />
<p align="justify"><br />
The basic mechanism of a kill switch is that engineered organisms survive normally until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which are small pieces of mRNA that bind ligands and modify translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/Hum</div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T19:22:19Z<p>LaurensDeBacker: </p>
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<h3 class="bg-yellow">Safety</h3><br />
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<p align="justify"><br />
It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p><br />
<p align="justify"><br />
Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.<br />
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<h3 class="bg-yellow">General Safety</h3><br />
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<p align = "justify"><br />
We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/><br />
We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.<br />
</div><br />
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<img src="https://static.igem.org/mediawiki/2013/6/66/Laurens_labo.png" alt="Laurens Labo"><br />
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<img src="https://static.igem.org/mediawiki/2013/f/ff/Ladybug_full.png" alt="some_text"><br />
<p><i>(Max Gibson)</i></p><br />
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<p align="justify"><br />
In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms requires extra safety conditions; physical measures are taken to separate the organism from the surrounding environment and vice versa.<br><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br><br />
The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of one bacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<h3>Legislation in Belgium & Europe</h3><br />
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<p align = "justify"><br />
In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
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<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3e/European_figure.jpg" alt="some_text"><br />
<p><i>(JRC - European Commission)</i></p><br />
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<h3>University of Leuven rules</h3><br />
<p align="justify"><br />
The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KUL's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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<h3 class="bg-yellow">Environmental safety</h3><br />
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<p align = "justify"><br />
If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
</div><br />
<br />
<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/Ladybug_tree.png"><br />
<p><i>(Norcal)</i></p><br />
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<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Bacterial safety</h3><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
</div><br />
<br />
<div class="span6"><br />
<p align = "justify"><br />
In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (Moe-Behrens ''et al.'', 2013), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br><br />
When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium therefore an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would chose for this method, we have to examine all the available kill switches and select the best one.</p><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>Auxotrophic mechanism</h3><br />
<div class="row-fluid"><br />
<br />
<div class="span9"><br />
<p align="justify"><br />
We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/><br />
However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png" alt="some_text"/><br/><br />
</div><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f2/Spray_antiaphid.png" alt="some_text"/><br />
</div><br />
<br />
<div class="span9"><br />
<h3>Kill Switch</h3><br />
<p align="justify"><br />
The basic mechanism of a kill switch is that engineered organisms survive normally until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which are small pieces of mRNA that bind ligands and modify translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/Hum</div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T19:09:04Z<p>LaurensDeBacker: </p>
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<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Safety</h3><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<p align="justify"><br />
It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p><br />
<p align="justify"><br />
Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">General Safety</h3><br />
</div><br />
</div><br />
<br />
<br />
<!-- TEKST --><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<div class="row-fluid"><br />
<div class="span9"><br />
<p align = "justify"><br />
We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/><br />
We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.<br />
</div><br />
<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/6/66/Laurens_labo.png" alt="Laurens Labo"><br />
</div><br />
<br />
</div><br />
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</div><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<a id="safety compounds"></a><br />
<div class="row-fluid"><br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/f/ff/Ladybug_full.png" alt="some_text"><br />
<p><i>(Max Gibson)</i></p><br />
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<div class="span9"><br />
<p align="justify"><br />
In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms requires extra safety conditions; physical measures are taken to separate the organism from the surrounding environment and vice versa.<br><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br><br />
The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of one bacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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<div class="row-fluid"><br />
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<p align = "justify"><br />
In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way, the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3e/European_figure.jpg" alt="some_text"><br />
<p><i>(JRC - European Commission)</i></p><br />
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</div><br />
</div><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>University of Leuven rules</h3><br />
<p align="justify"><br />
The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KUL's HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In these brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions about the possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
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<p align = "justify"><br />
If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
</div><br />
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<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/Ladybug_tree.png"><br />
<p><i>(Norcal)</i></p><br />
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<div id="header" class="row-fluid"><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
</div><br />
<br />
<div class="span6"><br />
<p align = "justify"><br />
In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (reference), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br><br />
When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium therefore an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would chose for this method, we have to examine all the available kill switches and select the best one.</p><br />
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<h3>Auxotrophic mechanism</h3><br />
<div class="row-fluid"><br />
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<div class="span9"><br />
<p align="justify"><br />
We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/><br />
However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png" alt="some_text"/><br/><br />
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<img src="https://static.igem.org/mediawiki/2013/f/f2/Spray_antiaphid.png" alt="some_text"/><br />
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<div class="span9"><br />
<h3>Kill Switch</h3><br />
<p align="justify"><br />
The basic mechanism of a kill switch is that engineered organisms survive normally until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which are small pieces of mRNA that bind ligands and modify translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/Hum</div>LaurensDeBackerhttp://2013.igem.org/Team:KU_Leuven/SafetyTeam:KU Leuven/Safety2013-10-04T18:52:35Z<p>LaurensDeBacker: </p>
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It is of course very important to stay safe. Here we have gathered information on how we stayed safe in our lab, what the rules of the government and the University of Leuven are and finally how we could keep our bacterium from spreading if it would actually be commercialised. We have also written an <a href="https://2013.igem.org/Team:KU_Leuven/Human_Practices/Ethics/Normative">ethical evaluation of our system</a> concerning some safety issues.</p><br />
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Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.<br />
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We have covered the safety aspect of our project in many ways. Within synthetic biology there are three main risks that have to be covered: exposition of the laboratory workers to the bacteria and chemicals, effects of a possible escape of the bacteria and the unintentional disturbance of the ecosystem. <br/><br />
We received extra safety training at the beginning of the project. On top of that we took courses regarding safety aspects, e.g. ‘Safe Microbiological Practices’. The safety training consisted of a summary of the important biosafety issues and the composition of the lab.<br />
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<img src="https://static.igem.org/mediawiki/2013/6/66/Laurens_labo.png" alt="Laurens Labo"><br />
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<img src="https://static.igem.org/mediawiki/2013/f/ff/Ladybug_full.png" alt="some_text"><br />
<p><i>(Max Gibson)</i></p><br />
</div><br />
<br />
<div class="span9"><br />
<p align="justify"><br />
In the past three months, we tried to add new branches to the pathways of <i>E. coli</i>. Our experiments were performed with non-pathogenic <i>E. coli</i> strains (BL21, DH5α, TOP10 and Rosetta™(DE3)pLysS), which belong to risk group 1 of the pathogenic organisms. Working with genetically modified organisms requires extra safety conditions; physical measures are taken to separate the organism from the surrounding environment and vice versa.<br><br />
Our project wants to protect plants and crops against aphid infestations and reduce the use of harmful insecticides. Our genetically modified bacteria will produce (E)-β-farnesene (EBF) and methyl salicylate (MeS), which are natural occurring substances. EBF repels aphids and MeS attracts ladybugs, one of their natural enemies. The low concentrations needed to be effective are not toxic for humans. No harmful effects are described for EBF at high concentrations either. Contrarily, pure MeS could cause harm when it is ingested due to its similarities with acetylsalicylic acid (aspirin) and can cause irritation after contact with eyes and skin. Nevertheless, it is typically used in small amounts as a flavouring agent in candy, or as fragrance oils due to its mint flavour and wintergreen fragrance. The two chemicals being produced by our BioBricks could cause allergic reactions (swelling, redness, ...), but serious reactions are very rare.<br><br />
The effect of our genetically modified bacteria on the environment is non-trivial and thorough field experiments must definitely be conducted. The maximum production rate of one bacterium has to be determined and scaling this model up to a whole field occupied by our bacteria will allow us to estimate the concentration in the surroundings. We can then determine the necessary parameters in order to minimise ecological disruption according to surrounding concentrations of MeS and EBF. We also thought about the interactions between aphids and ladybugs themselves. Aphids adapt readily when EBF is constantly present,therefore we included an oscillatory model. More information about this model can be found <a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator">here</a>. <br />
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</div><br />
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<div id="header" class="row-fluid"><br />
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<h3>Legislation in Belgium & Europe</h3><br />
<div class="row-fluid"><br />
<div class="span9"<br />
<p align = "justify"><br />
In Belgium, the whole research about genetically modified organisms (GMOs) is subjected to strict safety legislation. These laws must protect the people and the environment against the potential risks of the GMOs. The European legislation forms the foundation of the more extensive Belgian laws. Firstly, the use of GMOs in Europe requires an environmental licence. In Belgium, an approval for the research activity is required on top of that. This way the Belgian government controls all use of GMO’s and can assess risk analysis accurately. This is all recorded in the Decrees of the Flemish Government: Vlarem I and II section 51. The Belgian government describes four risk levels to which different restrictions are coupled. These restrictions are a combination of physical actions and safety regulations. For this purpose, the Belgian government appeals to the expertise of the Advisory Board of Biosafety (ABB). The ABB (section Biosafety and biotechnology of the Scientific Institute of Public Health) forms the scientific secretariat of the board. They evaluate the risk analysis in the licence request. At the KU Leuven, all the laboratories, including the iGEM lab, have an environmental licence and an approval for their activities.</p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/3/3e/European_figure.jpg" alt="some_text"><br />
<p><i>(JRC - European Commission)</i></p><br />
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</div><br />
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<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>University of Leuven rules</h3><br />
<p align="justify"><br />
The University of Leuven has its own biosafety rules in addition to the national biosafety regulations, where Marianne Schoukens acts as the central contact point for Health, Safety and Environment (HSE) issues. All members of our team have received training in Safe Microbiological Techniques as well as general and specific lab safety required by the University of Leuven. At the beginning of our project, all members of the iGEM team have received an update from the KU HSE department. Via liaison Iris Govaerts, we followed a small seminar and filled in an orientation checklist concerning health and safety training prior to the start of project laboratory work. <br><br />
The project we ultimately chose was the result of a whole series of brainstorm sessions. In this brainstorm sessions we talked about the feasibility of our ideas with our instructor (Dr. Ir. Ingmar Claes) and our coordinators (Prof.Dr. Johan Robben and Dr.Veerle De Wever). We also invited different PhD students, professors, ex-iGEMers and people from the industrial sector to our brainstorm sessions. These people have a lot more knowledge about the specific topics and could help us greatly with our decisions. Once the project was in progress, we also contacted a lot of people with questions and about possibilities. All these people had another view on our project and helped us taking into account different issues about the safety of our project: the molecules that our bacteria will produce, release into the environment, etc ...</p><br />
</div><br />
</div><br />
<br />
<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Environmental safety</h3><br />
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<div class="row-fluid"><br />
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<p align = "justify"><br />
If our project would be used around the country or in the whole world, many parameters would need to be determined. As stated earlier, the pheromones produced by the modified <i>E. coli</i> are not harmful for our health unless available in high concentrations. So when this modified <i>E. coli</i> would be spread across acres of land, the concentration of pheromones could attain a high level. The ceiling of this production would need to be determined to see whether it could be a harmful concentration for our health. Insects however do not need as high of a concentration as humans do to develop a reaction. Therefore the disruption of the ecosystem would need to be considered as well. As with most products released in the environment, its bioaccumulation needs to be at a minimum.</p><br />
</div><br />
<br />
<div class="span3"><br />
<img src="https://static.igem.org/mediawiki/2013/e/ec/Ladybug_tree.png"><br />
<p><i>(Norcal)</i></p><br />
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</div><br />
</div><br />
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<div id="header" class="row-fluid"><br />
<div class="span12"><br />
<h3 class="bg-yellow">Bacterial safety</h3><br />
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<img src="https://static.igem.org/mediawiki/2013/4/4c/Genetic_safeguard_strategies.jpg" alt="genetic safeguard strategies"><p align="justify"><b>Genetic safeguard strategies.</b><br/><br />
Recombinant DNA (bright green) is introduced into the host chromosome (white wavy lines). Two pathways for engineered auxotrophy (A,B) kill synthetic organisms (blue) once they lose access to a supplement (+) in a controlled environment. The supplement either (A) suppresses a toxic gene product (−) or (B) provides nutrition to compensate for a genetic deletion (red X). The induced lethality system (C) produces a toxic gene product (−) in response to an inducer (i) such as IPTG, sucrose, arabinose, or heat. (Moe-Behrens <i>et al.</i>, 2013)</p><br />
</div><br />
<br />
<div class="span6"><br />
<p align = "justify"><br />
In all our experiments, we used non-pathogenic <i>E. coli</i> strains. These strains are not harmful for humans, except when they are inhaled or swallowed. Nevertheless, they can cause irritation to the eyes and/or skin. The chemicals that are produced by our genetically modified <i>E. coli</i> strain are natural molecules and are not harmful for the environment. An accidental release of our bacteria into the environment could have unexpected effects on the ecological balance. As suggested in literature (reference), containment through engineered auxotrophy so that our genetically modified <i>E. coli</i> is unable to synthesise an essential compound required for their survival, is the most reliable method currently for biocontainment. We propose the use of a tryptophan auxotrophic bacterium.<br><br />
When tryptophan auxotrophic bacteria will be used in our project, we must provide tryptophan in their medium. This is possible when the bacteria grow together on a petri dish or another closed environment, but not when we want to spray the bacteria on the plants and crops. Therefore we designed two models. In one model the bacteria are grown in a contained environment, a plastic sticker (Design of Groningen iGEM 2012), where the auxotrophic mechanism can be used. This plastic sticker is composed of a plastic film with pores that are too small for the bacteria, but through which our volatiles (EBF and MeS) can escape.<br><br />
In the second model we have designed, the bacterium would be free in the environment and thus not contained in an enclosed container with medium therefore an auxotrophic mechanism would not be applicable. Here we turn to a ‘kill switch’ or induced lethality mechanism, which is less reliable because of spontaneous mutations that could inactivate or disable this ‘kill switch’. Different kill switches are already being used in the iGEM competition. When we would chose for this method, we have to examine all the available kill switches and select the best one.</p><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
<br />
<div class="row-fluid"><br />
<div class="span12 white"><br />
<h3>Auxotrophic mechanism</h3><br />
<div class="row-fluid"><br />
<br />
<div class="span9"><br />
<p align="justify"><br />
We, the KU Leuven iGEM team of 2013, propose the use of an auxotrophic mechanism as our main genetic safeguard, when we use our "sticker" or "oscillator" model. <br/><br />
The basic mechanism of auxotrophy is based on the fact that, since auxotrophic organisms are unable to synthesize an essential compound required for their survival, they rapidly die once they escape the controlled environment where the compound is supplied.<br/><br />
Already in 1987, Molin and colleagues designed a DNA cassette that functions as a conditional suicide system in any healthy bacterial strain. In the absence of an artificially supplied growth supplement, the cassette produced a toxic protein that damages bacterial cell membranes and kills the cells. Also, stochastic activation of this toxic component could be used to kill a predetermined fraction of cells per unit of time. This helps to tune the level of lethality so that an optimal level of bioproduction is achieved.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
This year, we present two models in which the use of an auxotrophic mechanism can be easily manifested, especially for the "sticker" or "oscillator" model.<br/><br />
However, auxotrophic mechanisms might be difficult to manage in our "spray" or "glucose" model. </p><br />
</div><br />
<br />
<div class="span3 greytext"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png" alt="some_text"/><br/><br />
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<div class="span9"><br />
<h3>Kill Switch</h3><br />
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The basic mechanism of a kill switch is that engineered organisms survive normally until an inducer signal (e.g., IPTG) is added, which kills the cells. This induced lethality could be used clean up synthetic microbe spills without harming other cells in the environment (Moe-Behrens <i>et al.</i>, 2013).<br/><br />
Inducible kill systems are not new to iGEM. There are already several constructs available in the database, such as the inducible BamHI system contributed by Berkeley in 2007 (<a href="http://parts.igem.org/Part:BBa_I716462">BBa_I716462</a>, tested by Lethbridge in 2011). Here, a <i>BamHI</i> gene was placed downstream of an arabinose-inducible promotor.<br/><br />
Unfortunately, the lethal gene is a central cause of safeguard failure. Spontaneous genetic mutations can both deactivate and activate lethal gene expression may worsen the failure of bio containment. As engineered cells are passaged in the laboratory, or as they propagate in large bioreactors, broken genetic safeguards can gradually accumulate in the population. If the utility of the bio containment mechanism is lost, then the synthetic organisms might survive in the environment after disposal or accidental release.(Moe-Behrens <i>et al.</i>, 2013)<br/><br />
Next, a lack of tight control is also a big problem within these systems. Leaky systems cannot be controlled strictly. Therefore, the Calgary team of 2012 added a riboswitch, which are small pieces of mRNA that bind ligands and modify translation of downstream genes; this way the kill switch can be more tightly controlled. For further details, we would gladly invite you to read the extensive literature study on the wiki of the <a href="https://2012.igem.org/Team:Calgary/Project/Hum</div>LaurensDeBacker