Team:KU Leuven/Project/Glucosemodel/EBF

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  <h3 class="bg-green">E-β-Farnesene</h3>
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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 alarm pheromone, released by almost all of the 4000 aphid species known thus far in response to the presence of predators (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, EBF can also cause long term effects : changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc. Moreover, natural aphid predators such as the ladybugs are attracted by EBF. <br/>
Hence, having our BanAphids produce EBF should help to repel aphids from our plant of choice. In the following sections, we will take you on a tour through the pathways that result in EBF, a general background of EBF synthase, the model and the genes, the wetlab work, and the biobricks we built for the EBF part. 
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  <h3 class="bg-green">The pathway to E-β-Farnesene</h3>
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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/>
FPP is the precursor of β-farnesene, that is produced by the building blocks, the molecules isopentenyl pyrophosphate (IPP) and its isomer dimethylallylpyrophosphate (DMAPP).<br/>
These precursors of farnesyl pyrophosphate can be produced by several metabolic pathways. Most prokaryotes use the non-mevalonate or DXP pathway, producing IPP starting from glyceraldehyde-3-phosphate and pyruvate. Eukaryotes, except for plants, exclusively use the mevalonate pathway, producing IPP starting from acetyl-CoA. Plants use both pathways.</p>
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    <img src="https://static.igem.org/mediawiki/2013/2/2a/Mevalonate_pathway.jpg" alt="Mevalonate Pathway">
    <p align="justify">Mevalonate pathway, showing the conversion of acetyl-CoA to general terpenoid precursor IPP and its isomer DMAPP. </p>
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    <img src="https://static.igem.org/mediawiki/2013/4/48/Non-Mevalonate.jpg" alt="Non-Mevalonate Pathway"/>
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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/>
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>
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    <p align="justify">Since FPP is such an important precursor, used for the biosynthesis of lots and lots of compounds, it might turn out that once we insert a plasmid containing the β-farnesene synthase gene, we 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 satisfy the EBF synthase enzyme we brought in.<br/>
A solution may be to implement plasmids which 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 for our uses. 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. </p>
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    <p align="justify">Since there are eight genes responsible for the mevalonate pathway, Martin <i>et al.</i> decided to split them up into two parts. A first plasmid named pMevT, 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 pMBIS, 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. A combined expression of their recombinant mevalonate pathway and the synthetic gene product (ADS in their case) resulted in greatly improved yields.</p>
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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. <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 by changing the amount of bacteria or the concentration of 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>
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  We cloned and expressed the EBF synthase gene in <i>E. coli</i>, to break up (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).<br/>
The enzyme prefers bivalent cations as cofactors; eg a Mg<sup>2+</sup> concentration of 5 mM should be beneficial for EBF function. The ideal pH for EBF synthase will be between 5.5-7.</p>
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Reaction.jpg" alt="reaction"/></center>
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The EBF construct we designed consists of constitutive promoter with a <i>lac</i> operator, the EBF synthase itself and a double terminator. 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 soil bacterium <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 the plant <i>Artemisia annua</i> (sweet wormwood) 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/>

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. 
For our construct, our first choice was a medium strength promoter with medium RBS (BBa_K608006); we nonetheless also made the construct with a strong promoter and RBS. We used BBa_B0015 for the double terminator. 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>
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  <h3>Gettin' the gene</h3>
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In the case of EBF synthase gene from <i>Streptomyces coelicolor</i>, we amplified this gene with the 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 we removed this site via site directed mutagenesis after transferring the gene into the iGEM pSB1C3 backbone. 
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  <h3>Cutting and pasting</h3>
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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 promoter or terminator as chassis, cut this open and inserted the gene / ... . the 3A assembly method was too difficult mostlikely because the size of promoter and terminator are so different compared to the gene insert. 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/>
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/>
For transformation, we used both chemically competent cells and electrocompetent cells. Electroporation had a higher efficiency when compared to heat shock transformation. </p>
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  <h3>Confirmation</h3>
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After we observed 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. Only the plasmids which succeeded in both controls were send for sequencing, the final confirmation.
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  <h3>gBlocks</h3>
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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. 
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For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult the page of our wetlab journal.
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<b>At the end of a trying summer, we finally made the following bricks.</b></p><ol>
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   <li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060001">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>
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   <li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060002">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>
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   <li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K106000">BBa_K1060008</a> This is an intermediate biobrick with EBF of <i>Artemisia annua</i> in front of a double terminator. </li>
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   <li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060009">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>
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   <li><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1060011">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>
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