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| <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/4/46/T--FJNU-China--2-PE_in_lid.png" style="width: 70%;"></p> | | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/4/46/T--FJNU-China--2-PE_in_lid.png" style="width: 70%;"></p> |
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− | <p> Taking biosafety into account, we added <span style="font-style:italic;">mazEF</span> to our circuit, which is a toxin-antitoxin module located on the <span style="font-style:italic;">Escherichia coli</span> chromosome and some of other bacteria, including pathogens. <span style="font-style:italic;">mazF</span> specifies for a stable toxin, mazF, and <span style="font-style:italic;">mazE</span> specifies for a labile antitoxin, mazE, which antagonizes <span style="font-style:italic;">mazF</span><span style="vertical-align:super;font-size:15px;">[1]</span>. <span style="font-style:italic;">mazF</span> is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and <span style="font-style:italic;">mazE</span> inhibits the RNase activity of <span style="font-style:italic;">mazF</span> by forming a complex.<span style="vertical-align:super;font-size:15px;">[2]</span>. In our project We used <span style="font-style:italic;">mazF</span> toxin protein as our killer to ensure biosafety. | + | <p> Taking biosafety into account, we added <span style="font-style:italic;">mazEF</span> to our circuit, which is a toxin-antitoxin module located on the <span style="font-style:italic;">Escherichia coli</span> chromosome and some of other bacteria, including pathogens. <span style="font-style:italic;">mazF</span> specifies for a stable toxin protein, and <span style="font-style:italic;">mazE</span> specifies for a labile antitoxin, which antagonizes toxin protein<span style="vertical-align:super;font-size:15px;">[1]</span>. <span style="font-style:italic;">mazF</span> is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and <span style="font-style:italic;">mazE</span> inhibits the RNase activity of <span style="font-style:italic;">mazF</span> by forming a complex.<span style="vertical-align:super;font-size:15px;">[2]</span>. In our project We used <span style="font-style:italic;">mazF</span> toxin protein as our killer to ensure biosafety. |
| <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/e/ee/T--FJNU-China--Description-mazEF.png" style="width: 70%;"></p> | | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/e/ee/T--FJNU-China--Description-mazEF.png" style="width: 70%;"></p> |
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| <h2 class="target"style="color: tomato;"> Bromidrosis</h2> | | <h2 class="target"style="color: tomato;"> Bromidrosis</h2> |
| <div class="col-md-12 col-xs-12"> | | <div class="col-md-12 col-xs-12"> |
− | <p> Aiming at another stink caused by bacteria, the bromidrosis is also an unpleasant smell which bothers people around. Two new circuits are applyied to the goal of controlling bromidrosis. </br> | + | <p> Aiming at another stink caused by bacteria, the bromidrosis is also an unpleasant smell which bothers people around. Two new circui |
− | When people sweat, there is specific salt concentration and temperature on the skin. We choose this feature as a starting point, designed and construced an engineered <span style="font-style:italic;">E.coli</span> strain with a logical gate to produce bacteriostatic PLA. Every basic component serves respective functions. <span style="font-style:italic;">dsrA</span>, a temperature-controlled promoter from <span style="font-style:italic;">E.coli</span>, is more active at 37 degree than 25 degree.<span style="vertical-align:super;font-size:15px;">[3]</span> The osmotically control promoter <span style="font-style:italic;">proU</span> shows sensitivity to the salt concentration from 0M to 0.3M.<span style="vertical-align:super;font-size:15px;">[4]</span> We used the global-regulate protein H-NS that could specifically bind to osmotically control promoter <span style="font-style:italic;">proU</span> as a specific repressor to inhibit the promoter downstream.<span style="vertical-align:super;font-size:15px;">[5]</span>
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− | </p>
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− | </div>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/b/ba/T--FJNU-China--on_skin.gif" style="width: 70%;">
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− | <p> When the circuits are spray on the skin, temperature reaches 37 degree, the repressor protein LacI will be expressed to inhibit <span style="font-style:italic;">PlacI</span> and the H-NS won’t be revealed. Thus, when people sweat, the Na<span style="vertical-align:super;">+</span> and Cl<span style="vertical-align:super;">-</span> will induce <span style="font-style:italic;">proU</span> resulting in the logical gate switching to “on” state.</p>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/7/79/T--FJNU-China--room-temperature.gif" style="width: 70%;">
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− | | + | |
− | <p> The circuit doesn’t need to be “on” state when it is at room temperature. When temperature stays below 37 ℃, <span style="font-style:italic;">dsrA</span> will not work. The function of PlacI BBa_R0011 has become a constitutive promoter. The global-regulate protein H-NS will be expressed to inhibit proU. Then the logical gate will be on “off” state. </br>
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− | We developed the 2-PE part as a switch that realizes the transformation from producing mode to suicide mode. <span style="font-style:italic;">TetO</span> and <span style="font-style:italic;">TetR</span> are common logic elements used in synthetic biology, linking with 2-PE and <span style="font-style:italic;">mazF</span>.<span style="vertical-align:super;font-size:15px;">[6][7]</span>
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− | </p>
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− | </div>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/b/bc/T--FJNU-China--producing-mode.gif" style="width: 70%;">
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− | <p> Without the induction of arabinose, <span style="font-style:italic;">TetR</span> and <span style="font-style:italic;">mazF</span> won’t be expressed, and production of 2-PE maintained at a normal level.</p>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/2/29/T--FJNU-China--killing-mode.gif" style="width: 70%;">
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− | <p> If users would like to stop 2-PE production, they only need to add a little arabinose. <span style="font-style:italic;">TetR</span> will be expressed to inhibit <span style="font-style:italic;">TetO</span>.The toxin protein <span style="font-style:italic;">mazF</span> would lead to the death of cell.</br>
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− | We currently use GFP and RFP to characterize the expression of our circuits. And we’re also trying to obtain more results. (Details can be found on our <a href="https://2018.igem.org/Team:FJNU-China/Result">Results</a> page.)
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− | </p>
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− | </div>
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− | | + | |
− | <div class="t4 col-md-12 col-xs-12">
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− | <hr>
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− | <h2 class="target" style="color: tomato;">Degradation</h2>
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− | <div class="col-md-12 col-xs-12">
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− | <p> We have known that the odorant acid E-3-methyl-2-hexenoicacid (E-3M2H) is an abundant and dominant human odorant. <span style="vertical-align:super;font-size:15px;">[8]</span> Therefore, we tried to eliminate such unpleasant axilla odors in an innovative method by degrading the odorant acid. Referring to potential production platform of n-butanol in <span style="font-style:italic;">E.coli</span><span style="vertical-align:super;font-size:15px;">[9]</span> , we designed a combined part to degrade E-3-methyl-2-hexenoicacid (E-3M2H).</p>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/8/8d/T--FJNU-China--degreding_enzyme.png" style="width: 70%;">
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− | | + | |
− | <p> This gene cluster expresses three enzymes, of which atoD and atoA from <span style="font-style:italic;">E.coli</span><span style=" and adhE2 from<span style="font-style:italic;">E.coli</span>Clostridium acetobutylicum ATCC 824<span style=" . The function of atoDA mediates reductive conversion of 3-methyl 2-hexenoic acid to 3-methyl-2-acetyl-CoA with acetyl-CoA as the CoA donor, for fatty acid activation and its subsequent degradation.<span style="vertical-align:super;font-size:15px;">[7]</span> AdhE2 catalyzes reduction of 3-methyl-2-acetyl-CoA to 3-methyl 2-hexenol at the expense of NADH.<span style="vertical-align:super;font-size:15px;">[10]</span> </p>
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− | <img class="img-responsive center-block" src="https://static.igem.org/mediawiki/2018/5/5a/T--FJNU-China--E-3M2H_pathway.jpg" style="width: 70%;">
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− | <hr>
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− | </div>
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− | <div>
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− | <hr>
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− | <h3>Reference</h3> [1]Engelberg-Kulka, H., et al. (2005). "mazEF: a chromosomal toxin-antitoxin module that triggers programmed cell death in bacteria." J Cell Sci 118(Pt 19): 4327-4332.</br>
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− | [2]Syed, M. A., et al. (2011). "The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system." J Bacteriol 193(5): 1122-1130. </br>
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− | [3] Repoila, F. and S. Gottesman (2003). "Temperature Sensing by the <span style="font-style:italic;">dsrA</span> Promoter." Journal of Bacteriology 185(22): 6609-6614.</br>
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− | [4] Kavalchuk, K., et al. (2012). "RNase III initiates rapid degradation of <span style="font-style:italic;">proU</span> mRNA upon hypo-osmotic stress in <span style="font-style:italic;">Escherichia coli</span>." RNA Biol 9(1): 98-109. </br>
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− | [5] Jan M. Lucht, Petra Dersch, Bettina Kempf and Erhard Bremer (1994) “Interactions of the Nucleoid-associated DNA-binding Protein H-NS with the Regulatory Region of the Osmotically Controlled <span style="font-style:italic;">proU</span> Operon of <span style="font-style:italic;">Escherichia coli</span>.” The JOUTNAL OF BIOLOGICAL CHEMISTRY 6578-6586.</br>
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− | [6]Soma, Y., Fujiwara, Y., Nakagawa, T., Tsuruno, K., & Hanai, T. (2017). Reconstruction of a metabolic regulatory network in <span style="font-style:italic;">Escherichia coli</span> for purposeful switching from cell growth mode to production mode in direct GABA fermentation from glucose. Metabolic Engineering, 43(August), 54–63.</br>
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− | [7]Soma, Y., Tsuruno, K., Wada, M., Yokota, A., Hanai, T., 2014. Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch. Metab. Eng. 23, 175–184.</br>
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− | [8] Andreas Natsch*. What Makes Us Smell: The Biochemistry of Body Odour and the Design of New Deodorant Ingredients. KGF-SCS Industrial Investigator Award 2014</br>
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− | [9] Saini M, Chen M H, Chiang C J, et al. Potential production platform of n-butanol in Escherichia coli[J]. Metabolic Engineering, 2015, 27:76.</br>
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− | [10] Anu Jose Mattam and Syed Shams Yazdan. Engineering E. coli strain for conversion of short chain fatty acids to bioalcohols. Biotechnol Biofuels. 2013; 6: 128.</br>
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