Http://2013.igem.org/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 present you the insight of the E-β-farnesene (aka EBF) project. EBF is the most universal alarm pheromone that is released by almost all the 4000 aphid species in response to predation or other disturbance, and the beneficial consequence among aphids due to EBF is presumed to be allowing the population taking evasive action after perception of EBF, therefore the successful survival rate will be increased. In addition to the short term repelling effect, EBF can also cause the change in aphid’s development, fecundity, survival when introduced to different growth stages of aphid in a long term. Moreover, the natural predators of aphid such as ladybug can recognize EBF as well; this will lead to the attraction of ladybug.<br/>
Hence, we chose EBF as a major part in our design to perform the act of repelling the aphids away. In the following sections, we will bring you on a tour of the general background, the model and the genes, the wetlab work, and the biobricks we built. 
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  <h3 class="bg-green">General background of the enzyme</h3>
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  Herein, we incorporated EBF synthase into the metabolic pathway of E.coli, with which, (2E,6E)-farnesyl diphosphate will be converted into (E)-beta-farnesene (EBF) and diphosphate as depicted in the reaction below.<br/>
The enzyme’s activity will be achieved the maximum with the present of Mg<sup>2+</sup> with the concentration of 5mM. The ideal pH for EBF synthase will fall in the range of 5.5-7.
For the <i>Artemisia annua</i> protein the K<sub>M</sub> is 0.0021mM, Kcat/K<sub>M</sub>=4.5, turnover number 0.0095s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 and the turnover number 0.019s<sup>-1<sup>. </p>
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Reaction.jpg" alt="reaction"/></center>
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  <h3 class="bg-green">General background of the enzyme</h3>
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  Herein, we incorporated EBF synthase into the metabolic pathway of E.coli, with which, (2E,6E)-farnesyl diphosphate will be converted into (E)-beta-farnesene (EBF) and diphosphate as depicted in the reaction below.<br/>
The enzyme’s activity will be achieved the maximum with the present of Mg<sup>2+</sup> with the concentration of 5mM. The ideal pH for EBF synthase will fall in the range of 5.5-7.
For the <i>Artemisia annua</i> protein the K<sub>M</sub> is 0.0021mM, Kcat/K<sub>M</sub>=4.5, turnover number 0.0095s<sup>-1</sup>. For the <i>Streptomyces coelicolor</i> protein the K<sub>M</sub> is 0.0168 and the turnover number 0.019s<sup>-1<sup>. </p>
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Reaction.jpg" alt="reaction"/></center>
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The construct we designed for EBF project consists of constitutive promoter + lac operator + EBF synthase + double terminator (refer to project description). There are several organisms contain EBF synthase gene, and we obtained two sources of this gene, one is <i>Streptomyces coelicolor</i> from Centre of Microbial and Plant Genetics of KUL, and the other one is <i>Artemisia annua<i> (sweet wormwood) from a research group in University Kalmar. However, the EBF synthase from <i>Streptomyces coelicolor</i> is a bifunctional enzyme (containing albaflavenone synthase activity), so the following build up of the final construct is based on EBF synthase gene from Artemisia annua. <br/>
For the constitutive promoter, we chose a medium promoter with medium RBS (BBa_K608006), because the constitutive expression of EBF does not need to be strong. For the double terminator, we decided to use BBa_B0015. The lac operator in front of the EBF synthase gene will play a role of switching the transcription of EBF synthase gene.</p>
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  <h3>Gettin' the gene</h3>
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To get the two sources of the target gene, we exploited different methods. In the case of EBF synthase gene from Streptomyces coelicolor, we got this gene with the help of colony PCR. In the story of EBF synthase gene from Artemisia annua, we received the gene in the pET28 vector from the research group in university of Kalmar, but there is an additional EcoRI restriction site in the gene, which will conflict with the standard iGEM cloning work, thus we performed site specific mutation to get rid of this restriction site after transferring the gene into pSB1C3 backbone. 
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  <h3>Cutting and pasting</h3>
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After obtaining the target gene in the standard pSB1C3 backbone, we started our cloning work. The general concept we adapted is cutting the vector of promoter or terminator open, in which case the promoter or terminator is at the end of the linearized vector, then ligate the insert into the vector. The reason why we undertake this strategy is because the size of promoter and terminator is too small, the possibility of false positive will be enhanced quite a lot if we use the 3A assembly method. In the situation of ligating the insert in front of the double terminator, we cut the vector of double terminator with EcoRI and XbaI restriction sites, cut the insert with EcoRI and SpeI restriction site. On the other hand, the promoter vector is cutted with SpeI and PstI restriction sites, and the insert is cutted with XbaI and PstI restriction sites. <br/>
In the terms of ligation, we performed in two ways in parallel. The first one is ligate for 20 minutes at 16℃, in comparison, the second ligation of the same products is conducted in 16℃ overnight. </p>
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  <h3>Cutting and pasting</h3>
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After obtaining the target gene in the standard pSB1C3 backbone, we started our cloning work. The general concept we adapted is cutting the vector of promoter or terminator open, in which case the promoter or terminator is at the end of the linearized vector, then ligate the insert into the vector. The reason why we undertake this strategy is because the size of promoter and terminator is too small, the possibility of false positive will be enhanced quite a lot if we use the 3A assembly method. In the situation of ligating the insert in front of the double terminator, we cut the vector of double terminator with EcoRI and XbaI restriction sites, cut the insert with EcoRI and SpeI restriction site. On the other hand, the promoter vector is cutted with SpeI and PstI restriction sites, and the insert is cutted with XbaI and PstI restriction sites. <br/>
In the terms of ligation, we performed in two ways in parallel. The first one is ligate for 20 minutes at 16℃, in comparison, the second ligation of the same products is conducted in 16℃ overnight.<br/>
Here comes transformation after ligation, we have both chemical competent cells and electrocompetent cells, there is no big difference between them, the only thing is electroporation has higher efficiency when compared to heat shock transformation. </p>
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  <h3>Confirmation</h3>
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As soon as we observe colonies after transformation, we need to confirm the products. The first step we do is usually a colony PCR to check if the insert is in the vector; however the colony PCR does not work every time. If it works, we will select the good ones to inoculate, otherwise randomly select some colonies to do the inoculation. The plasmid extraction is done on the overnight inoculated culture, and then we can confirm the size by digestion the plasmid. Only if the sample preceded these two confirmations, we will send the good ones to sequence, which will be the final confirmation. 
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  <h3>G-blocks</h3>
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Meanwhile, we also built the construct with lac operator in between the promoter and gene with gBlocks. We designed the gBlocks and assembled them together, followed by ligating the insert into pSB1C3 backbone. The positive colonies also need to go through three confirmation steps mentioned above before we conclude we made them. 
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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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  <h3 class="bg-green">Our Bricks</h3>
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Of course we met a lot of difficulties during the cloning work, but we kept trying different ways to overcome the obstacles, and finally we made the following bricks.<ol>
<li>BBa_K1060001 This is a coding biobrick with the insert length of 1386bp, it is EBF synthase gene from Streptomyces coelicolor in pSB1C3 backbone.</li>
<li>BBa_K1060002 This is another coding biobrick with the insert length of 1725bp, it is EBF synthase gene from Artemisia annua in pSB1C3 backbone.</li>
<li>BBa_K1060008 This is an intermediate biobrick with EBF of Artemisia annua in front of double terminator. </li>
<li>BBa_K1060009 This is a generator biobrick with the insert length of 1924bp, it is medium constitutive expression of EBF synthase from Artemisia annua, in the pSB1C3 backbone.</li>
<li>BBa_K1060014 This is another generator biobrick with the insert length of 1923bp, it is strong constitutive expression of EBF synthase from <i>Artemisia annua</i>, in the pSB1C3 backbone.</li>
<li>BBa_K1060011 This is a generator biobrick with the insert length of 1965bp, it is medium constitutive expression of EBF synthase from <i>Artemisia annua</i> with lac operator after the promoter.</li></ol></p>
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