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<h1>Basic Part</h1>
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<a href="#wrapper">
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<div style='padding-top: 100px;'><h1 id="wrapper"><img src="https://static.igem.org/mediawiki/2018/2/27/T--Kyoto--workflow.png" width="30%"></div></h1>
  
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  <h5>Rev Protein</h5>
 
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  <p>For this year’s iGEM competition, we have chosen to submit Rev protein for the award of <a href="http://parts.igem.org/Part:BBa_K2403000">the Best Basic part (BBa_K2403000)</a>. In eukaryotic cells mature mRNA is exported by Tap/p15 (Mex67p/ Mtr2p in yeast) from the nucleus into the cytoplasm for translation into protein. However, in synthetic biology, other types of RNA with complex structures are frequently designed and used. If RNA with complex structure is expressed in a eukaryotic cell, Tap/p15 cannot export it effectively, and the RNA will remain in the nucleus. To solve this problem, we developed parts for the HIV Rev protein and associated cis-acting RRE (rev response element) for nuclear export of artificial RNA. iGEMers can export their RNA sequences containing an RRE to the cytoplasm if they express Rev protein simultaneously. </p>
 
  
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/d/de/Kyoto_fig6b.jpeg" width="60%"></p>
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<div class="box27">
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    <span class="box-title"><font face="Segoe UI">Table of contents</font></span>
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  <h5>How Rev works</h5>
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    <ul class="index1">
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            <li><a href="#res1">1) Creation of KO yeast strains</a></li>
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            <li><a href="#res2">2) Plasmid construction</a></li>
 +
            <li><a href="#res3">3) Assesment of halotorelance</a></li>
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            <li><a href="#res4">4) Assesment of the amount of absorbing Na+</a></li>
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            <li><a href="#res5">5) Assesment of aggregation</a></li>
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</ul>
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  <p>Rev recruits CRM1, a principal nuclear export factor in cells which mediates export of cellular RNAs including snRNA and all 4 ribosomal RNAs to the cytoplasm.</p>
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<h5 id="res1">1)Creation of KO yeast strains</h5>
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<p class="description">At a first step, we worked on creation of KO yeast strains which uptake more sodium and therefore show salt-sensitivity. It is because we need to use salt sensitive yeast strains in order to assess functions of proteins which contribute salt torelance, and also Na+を外に漏らさずため込ませたかったからだ。
 +
作り方は<a href="https://2018.igem.org/Team:Kyoto/SpecialMethods"><font color=#000000;>Special protocol</font></a>を見てください。
 +
Based on Aachen 2017's result, we created ΔNHA1, ΔENA1ΔNHA1,ΔENA1 at first, and 実験を進めるにあたって東北大学の魚住先生にいただいたG19株(ΔENA1,2,3,4)がよく塩を吸収し、高い塩感受性を示すことがわかり、ENA1だけでなく同じタンデムにあるENA2,3,4もノックアウトする方がいいことがわかった。またNHA1をノックアウトすることも塩吸収に貢献していたため、最後にΔENA1,2,5ΔNHA1も作成しました。以下が私たちが作成した変異株です。実験においては、上記のG19株と、渡部先生にいただいた醤油酵母も用いた。
 +
<ul class="strain">
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<li>・ΔNHA1</li>
 +
<li>・ΔENA1ΔNHA1</li>
 +
<li>・ΔENA1</li>
 +
<li>・ΔENA1,2,5ΔNHA1</li>
 +
</ul></p>
  
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  <h5>Key advantage</h5>
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<h5 id="res2">2)Plasmid construction</h5>
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+
<p class="description">次に、私たちはプラスミドのコンストラクションを行いました。デザインページにあるように、塩耐性のためにMangrin, ZrGPD1,ZrFPS1を、塩の回収のためにAtHKT1,AVP1, AtNHXS1, SseNHX1の作成をしました。
−
  <p>Rev protein makes it possible to export complex RNA structures from the nucleus to the cytoplasm, which otherwise cannot be achieved effectively.
+
 
 +
 
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<p><i>text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text  </i> </p>
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<p class="pic"><img src="https://static.igem.org/mediawiki/2018/c/ca/T--Kyoto--sample--image.jpg" width="60%">
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<p class="caption"><b>text text text text text text text text text text text text text text text</b> text text text text text text text text text text text text text text text text text text text text text text text text text text text text text <br>
 +
 
 +
<p>text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text </p>
 +
 
 +
 
 +
<h5 id="res3">3)Assesment of halotorelance</h5>
 +
 
 +
 
 +
<h5 id="res4">4)Assesment of the amount of absorbing Na+</h5>
 +
 
 +
 
 +
<h5 id="res5">5)Assesment of aggregation</h5>
 +
 
 +
 
 +
<p class="pic"><img src="https://static.igem.org/mediawiki/2018/c/ca/T--Kyoto--sample--image.jpg" width="60%"></p>
 +
 
 +
<p class="pic"><img src="https://static.igem.org/mediawiki/2018/c/ca/T--Kyoto--sample--image.jpg" width="60%"></p>
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<p class="caption">
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<b>Figure 2-a</b> caption caption caption caption caption<br>
 +
 
 +
  <h6>Reference</h6>
 +
      <ul class="reference">
 +
<li>[1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in <i>Bursaphelenchus xylophilus</i> causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.<li>
 +
<li>[2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.</li>
 +
<li>[3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.</li>
 +
<li>[4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.</li>
 +
<li>[5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.</li>
 
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Latest revision as of 16:41, 17 October 2018

Team:Kyoto/Project - 2018.igem.org

Table of contents
1)Creation of KO yeast strains

At a first step, we worked on creation of KO yeast strains which uptake more sodium and therefore show salt-sensitivity. It is because we need to use salt sensitive yeast strains in order to assess functions of proteins which contribute salt torelance, and also Na+を外に漏らさずため込ませたかったからだ。 作り方はSpecial protocolを見てください。 Based on Aachen 2017's result, we created ΔNHA1, ΔENA1ΔNHA1,ΔENA1 at first, and 実験を進めるにあたって東北大学の魚住先生にいただいたG19株(ΔENA1,2,3,4)がよく塩を吸収し、高い塩感受性を示すことがわかり、ENA1だけでなく同じタンデムにあるENA2,3,4もノックアウトする方がいいことがわかった。またNHA1をノックアウトすることも塩吸収に貢献していたため、最後にΔENA1,2,5ΔNHA1も作成しました。以下が私たちが作成した変異株です。実験においては、上記のG19株と、渡部先生にいただいた醤油酵母も用いた。

  • ・ΔNHA1
  • ・ΔENA1ΔNHA1
  • ・ΔENA1
  • ・ΔENA1,2,5ΔNHA1

2)Plasmid construction

次に、私たちはプラスミドのコンストラクションを行いました。デザインページにあるように、塩耐性のためにMangrin, ZrGPD1,ZrFPS1を、塩の回収のためにAtHKT1,AVP1, AtNHXS1, SseNHX1の作成をしました。

text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text

text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text

text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text text

3)Assesment of halotorelance
4)Assesment of the amount of absorbing Na+
5)Assesment of aggregation

Figure 2-a caption caption caption caption caption

Reference
  • [1] X. rong Wang, X. Cheng, Y. dong Li, J. ai Zhang, Z. fen Zhang, and H. rong Wu, “Cloning arginine kinase gene and its RNAi in Bursaphelenchus xylophilus causing pine wilt disease,” Eur. J. Plant Pathol., vol. 134, no. 3, pp. 521–532, 2012.
  • [2] A. Sigova, N. Rhind, and P. D. Zamore, “A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe,” genes Dev., 2004.
  • [3] R. Esteban and R. B. Wickner, “A new non-mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains.,” Genetics, 1987.
  • [4] M. T. B. Sloan, Katherine E, Pierre-Emmanuel Gleizes, “Nucleocytoplasmic Transport of RNAs and RNA–Protein Complexes,” J. Mol. Biol., vol. 428, no. 10, pp. 2040–2059, 2016.
  • [5] V. W. Pollard and M. H. Malim, “the Hiv-1 Rev Protein,” Annu. Rev. Microbiol., vol. 52, no. 1, pp. 491–532, 1998.