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| <p class="normal_text">When you were very young, before going to sleep, have you ever asked your mother, “Mommy, read some picture books to me, please.”? You may have been read it aloud at least once. <br> | | <p class="normal_text">When you were very young, before going to sleep, have you ever asked your mother, “Mommy, read some picture books to me, please.”? You may have been read it aloud at least once. <br> |
| <p class="normal_text">Most stories for generations have lessons. The lessons from Snow White story are mainly the following two points.</p> | | <p class="normal_text">Most stories for generations have lessons. The lessons from Snow White story are mainly the following two points.</p> |
− | Ⅰ It is not good to show off or to envy someone.<br> | + | <p class="normal_text">Ⅰ Do not show off or envy someone.</p> |
− | Ⅱ Do not trust unfamiliar people blindly. <br> | + | <p class="normal_text">Ⅱ Do not trust unfamiliar people blindly. </p> |
| <p class="normal_text">It is not enough to be just friendly. A beautiful mind needs blessing with not only tenderness and richness but also a strong side that controls one’s feelings.</p> | | <p class="normal_text">It is not enough to be just friendly. A beautiful mind needs blessing with not only tenderness and richness but also a strong side that controls one’s feelings.</p> |
| <p class="normal_text">In our project, we will comprehensively evaluate Snow White story from the perspectives of two beauty, physical beauty and inner beauty.</p> | | <p class="normal_text">In our project, we will comprehensively evaluate Snow White story from the perspectives of two beauty, physical beauty and inner beauty.</p> |
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| <p class="normal_text">Then, she opened her eyes!</p><br> | | <p class="normal_text">Then, she opened her eyes!</p><br> |
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− | <p class="normal_text">This dislodged from Snow White’s throat the piece of poisoned apple that she had bitten off.</p><br><br> | + | <p class="normal_text">because this action dislodged from Snow White’s throat the piece of poisoned apple that she had bitten off.</p><br><br> |
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| <p class="normal_text">THE END</p><br><br><br><br> | | <p class="normal_text">THE END</p><br><br><br><br> |
− | | + | <p class="normal_text">In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP and GFP.</p> |
| + | <p class="normal_text">We use the graph shown below to evaluate. The vertical axis of the graph shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time.</p> |
| + | <p class="normal_text">No matter how beautiful one’s physical appearance is, the one cannot be a real beauty unless has a beautiful mind. We will show you which woman has a real beauty, Snow White or the Queen. </p> |
| <p class="normal_text">The beauty is defined as follows in this project.<br> | | <p class="normal_text">The beauty is defined as follows in this project.<br> |
| <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Beauty = physical beauty + inner beauty</span> | | <p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Beauty = physical beauty + inner beauty</span> |
| </p><br> | | </p><br> |
− | <p class="normal_text">In our project, we evaluate the real beauty of Snow White and the Queen with the fluorescence intensities of RFP and GFP.</p> | + | |
− | <p class="normal_text">We use the graph shown below to evaluate. The vertical axis of the graph shows the beauty of Snow White and the Queen, and the horizontal axis shows the elapsed time.</p>
| + | |
− | <p class="normal_text">No matter how beautiful one’s physical appearance is, the one cannot be a real beauty unless has a beautiful mind. We will show you which woman has a real beauty, Snow White or the Queen. </p>
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| <div id="system_contents" class="container_contents"> | | <div id="system_contents" class="container_contents"> |
| <p class="normal_text"> Before introduction of our genetic circuit’s design, we will introduce a system that form the basis of our project. | | <p class="normal_text"> Before introduction of our genetic circuit’s design, we will introduce a system that form the basis of our project. |
− | <p class="normal_text">They are TA system. | + | They are TA system. |
| </p> | | </p> |
| <div id="sys1_header"> | | <div id="sys1_header"> |
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| <div id="sys1"> | | <div id="sys1"> |
| <div id="sys1_contents"> | | <div id="sys1_contents"> |
− | <p class="normal_text">A toxin-antitoxin system is composed of two or more cognate genes that encode toxins and antitoxins. Toxins are proteins, whereas antitoxins are either proteins or non-coding RNAs. [1] Many prokaryotes have toxin-antitoxin systems, and organisms often have them in multiple copies. [2][3] Changes in the physiological conditions, such as stress conditions or viral infection leads to the antitoxin degradation, and proteases degrade many antitoxins. Unleashed toxin proteins impede or alter cellular processes including translation, cell division, DNA replication, ATP synthesis, mRNA stability or cell wall synthesis and lead to dormancy. This dormant state probably enables the bacteria to survive in unfavorable conditions. | + | <p class="normal_text">A toxin-antitoxin system is composed of two or more cognate genes that encode toxins and antitoxins. Toxins are proteins, whereas antitoxins are either proteins or non-coding RNAs. Many prokaryotes have toxin-antitoxin systems, and organisms often have them in multiple copies. Changes in the physiological conditions, such as stress conditions or viral infection leads to the antitoxin degradation, and proteases degrade many antitoxins. Unleashed toxin proteins impede or alter cellular processes including translation, cell division, DNA replication, ATP synthesis, mRNA stability or cell wall synthesis and lead to dormancy. This dormant state probably enables the bacteria to survive in unfavorable conditions. |
| In general, toxin is more stable than antitoxin, but antitoxin is expressed to a higher level.<br><br> | | In general, toxin is more stable than antitoxin, but antitoxin is expressed to a higher level.<br><br> |
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− | <div align="center"><p class="normal_text">First toxin‐antitoxin: MazF / MazE</p><br></div> | + | <div align="center"><p class="normal_text">First toxin‐antitoxin: MazF / MazE</p></div> |
− | <p class="normal_text">MazF is a toxin protein, and MazE is its cognate antitoxin protein. MazF is a ribosome-independent endoribonuclease. This endoribonuclease activity leads to bacterial growth arrest. MazE and MazF form homodimers, and MazF dimer cleaves mRNAs at ACA sites. [4] One MazE dimer binds to two MazF dimers, thereby inactivates endoribonuclease activity of MazF dimer. [5] MazE is labile, subjected to degradation by ClpAP protease, whereas MazF is more stable. [6]<br><br> | + | <p class="normal_text">MazF is a toxin protein, and MazE is its cognate antitoxin protein. MazF is a ribosome-independent endoribonuclease. This endoribonuclease activity leads to bacterial growth arrest. MazE and MazF form homodimers, and MazF dimer cleaves mRNAs at ACA sites. One MazE dimer binds to two MazF dimers, thereby inactivates endoribonuclease activity of MazF dimer. MazE is labile, subjected to degradation by ClpAP protease, whereas MazF is more stable. <br><br> |
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| </p> | | </p> |
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− | <div align="center"><p class="normal_text">Second toxin‐antitoxin: YafO / YafN</p><br></div> | + | <div align="center"><p class="normal_text">Second toxin‐antitoxin: YafO / YafN</p></div> |
− | <p class="normal_text">YafO is a toxin protein, and YafN is its cognate antitoxin protein. [7] YafO is a ribosome-associated mRNA interferase that cleaves mRNAs’ downstream 11-13 bases of the translation initiation sites. When 70S ribosome is dissociated, YafO associates with 50S ribosome subunit and gets endoribonuclease activity. YafO and YafN forms a complex Via binding with YafN, YafO is neutralized. [8] YafN is labile, subjected to degradation by Lon protease, whereas YafO is more stable [9].<br><br> | + | <p class="normal_text">YafO is a toxin protein, and YafN is its cognate antitoxin protein. YafO is a ribosome-associated mRNA interferase that cleaves mRNAs’ downstream 11‐13 bases of the translation initiation sites. When 70S ribosome is dissociated, YafO associates with 50S ribosome subunit and gets endoribonuclease activity. YafO and YafN forms a complex Via binding with YafN, YafO is neutralized. YafN is labile, subjected to degradation by Lon protease, whereas YafO is more stable.<br><br> |
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| </p> | | </p> |
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| <div id="sys2_header"> | | <div id="sys2_header"> |
− | <h3><span>The art of MazF / MazE </span></h3> | + | <h3><span>3.2 The art of MazF / MazE </span></h3> |
| </div><!-- /_header --> | | </div><!-- /_header --> |
| <div id="sys2"> | | <div id="sys2"> |
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| Then, she opened her eyes!<br></font> | | Then, she opened her eyes!<br></font> |
| <br> | | <br> |
− | The Prince <span style="font-style : italic">coli</span> which has received 3OC12HSL expresses AmiE. In general, AmiE degrades HSL with more than 7 carbons. This leads to the degradation of the Poisoned Apple 3OC12HSL.
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− | When the Poisoned Apple is degraded, MazF expression is inhibited in Snow White, and gradually MazF function is counteracted by MazE. Then, translation restarts in Snow White <span style="font-style : italic">coli</span>.
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| <br> | | <br> |
− | </p><br>
| |
| </div> | | </div> |
| </div> | | </div> |
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| <p class="normal_text">5.1 Cold inducible promoter functions at 18°C in Scene 1<br> | | <p class="normal_text">5.1 Cold inducible promoter functions at 18°C in Scene 1<br> |
| It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. This story never begins unless the Magic Mirror answers the Queen’s question.<br><br> | | It is a cold inducible promoter (called Pcold commonly) that plays a most important role in this Scene. This story never begins unless the Magic Mirror answers the Queen’s question.<br><br> |
− | The experiment was conducted with BBa_1949001. We cultivated each sample at 18°C and 37°C and measured the GFP / Turbidity with a plate reader. The experimental result showed that samples cultured at 18°C had higher fluorescence intensity of GFP than those cultured at 37°C. | + | The experiment was conducted with BBa_1949001. We cultivated each sample at 18°C and 37°C and measured the RFU of GFP / Turbidity with a plate reader. The experimental result showed that samples cultured at 18°C had higher fluorescence intensity of GFP than those cultured at 37°C. |
| Thus, we found that the story begins by lowering the culture temperature. | | Thus, we found that the story begins by lowering the culture temperature. |
| <br><br></p> | | <br><br></p> |
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| <div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"></div> | | <div align="center"><img src="https://static.igem.org/mediawiki/2016/0/00/T--Tokyo_Tech--assay_ahl_reporter.png" height ="200"></div> |
| <p class="normal_text">Although C12 is added to Plux, we found that the promoter activity can be seen. In addition, the graph shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when added C4.</p> | | <p class="normal_text">Although C12 is added to Plux, we found that the promoter activity can be seen. In addition, the graph shows that Prhl has a large leak, and the Prhl activity can be hardly seen compared to the others when added C4.</p> |
− | <p class="normal_text">The Queen <span style="font-style : italic">coli</span>, which has received C4HSL, produces LasI and MazF.LasI produces a signaling molecule, 3OC12HSL which is the Poisoned Apple.
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− | Additionally, produced MazF inhibits the translation in the Queen. If the translation is inhibited the moment MazF is expressed, sufficient amount of LasI will not be produced, and neither will 3OC12HSL. Then the Queen will fail the assassination of Snow White.</p>
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| <br> | | <br> |
| </p><br> | | </p><br> |