Difference between revisions of "Team:Kyoto/Workflow"

 
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<div id="jump"><a href="#wrapper"><img src="https://static.igem.org/mediawiki/2018/b/b5/T--Kyoto--UpButton.png"></a></div>
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  <h1>Workflow(未)</h1><br><p>締め切り：　原稿担当：童</p>
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<a href="#wrapper">
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<img src="https://static.igem.org/mediawiki/2018/1/11/T--Kyoto--upbotton.jpg"></a></div>
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<div id="BACKGROUND">
  
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    <h5>Table of contents</h5>
 
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  <ul class="result">
 
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      <li>Na+ absorbing work</li>
 
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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>
 
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      <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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      <li><a href="#res6">6) </a></li>
 
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  </ul>
 
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<h5 id="res1">1) Creation of KO yeast strains</h5>
 
  
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<p class="pic"><img src="https://static.igem.org/mediawiki/2018/c/ca/T--Kyoto--sample--image.jpg" width="50%">
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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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<p class="caption"><b>Figure 1-a</b>
 
  
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<i>S. cerevisiae</i> cells.<br>  
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<div class="box27">
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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><br>
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    <span class="box-title"><font face="Segoe UI">Table of contents</font></span>
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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>
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            <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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</div>
  
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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。
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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">
 +
<li>・ΔNHA1</li>
 +
<li>・ΔENA1ΔNHA1</li>
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<li>・ΔENA1</li>
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<li>・ΔENA1,2,5ΔNHA1</li>
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</ul></p>
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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。
  
  
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<h5 id="res3">3)Assesment of halotorelance</h5>
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<h5 id="res4">4)Assesment of the amount of absorbing Na+</h5>
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<h5 id="res5">5)Assesment of aggregation</h5>
  
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/8e/Flo2.png" width="45%">
 
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<p class="caption"><b>Figure 2-e</b> Time course of the rate of eGFP(+) nematodes.<br>
 
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Nematodes were grown on eGFP(+) yeast and examined by fluorescence microscopy at the indicated time. (n=18)
 
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</p>
 
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<br>
 
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<h5 id="res3">3) Choose dsRNA</h5>
 
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<p>In order to kill <I>B. xylophilus</I>, it is necessary to efficiently knock-down genes essential for growth. Examining the literature, we found a paper that succeeded in knocking down essential genes and reducing the survival rate by submerging <I>B. xylophilus</I> in high concentrations of dsRNA for a certain period of time (soaking RNAi) [1]. In this paper, the target was mRNA of arginine kinase AK1 which was an essential gene expressed in the intestines and RNAi of AK1 showed a fatal effect also in C. elegans. AK1 is an invertebrate-specific key enzyme of energy metabolism so it is often used as a target for development of invertebrate-specific inhibitors. It was a promising candidate for dsRNA expressed in yeast.
 
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In order to select the most ideal target, we prepared dsRNA of several target mRNAs including AK1 in vitro and tried soaking RNAi. We obtained target sequence from a public database, designed oligos, and cloned genes by RT-PCR. At this time, we put the T7 promoter on both ends of the DNA so that dsRNA was synthesized by in vitro transcription. After transcription, association of dsRNA was induced by an annealing operation, and the template was removed with DNase. We confirmed the dsRNA finally obtained by electrophoresis (Figure 3-a).
 
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</p>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/79/Kyoto_fig3a.png" width="45%">
 
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<p class="caption"><b>Figure 3-a</b> In vitro synthesized dsRNAs for soaking RNAi experiments.<br>
 
−
M DNA size marker, λSty I<br>
 
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1 dsAK-2 (692-bp)<br>
 
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2 dsEef-1g (528-bp)<br>
 
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3 dsAK-1 (449-bp)<br>
 
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4 dsAsb (559-bp)<br>
 
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5 ds14-3-3zeta (534-bp)<br>
 
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6 dsTropomyosin (532-bp)<br>
 
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7 ds14-3-3 protein (610-bp)<br>
 
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8 dsGFP (649-bp)
 
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</p>
 
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</p>
 
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<br>
 
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<p>We prepared these RNAs, adjusted to a concentration of 2 μg /μL, and tried soaking RNAi The results is shown in the figure (Figure 3-b). As is clearly shown, we could not see the phenotype due to the introduction of dsRNA which was inconsistent with the previously reported example. As a result of contacting several <I>B. xylophilus</I> researchers and gathering information, it turned out that even several Japanese researchers have attempted to reproduce <I>B. xylophilus</I> soaking RNAi, but no group was able to observe a clear effect. The reason may be that soaking RNAi of <I>B. xylophilus</I> contains technically unstable steps. Alternatively, since <I>B. xylophilus</I> used this time is derived from wild nematodes collected from the field, there may be a difference between the strain we used and nematodes in the publication where soaking RNAi was effective. </p>
 
−
<p>Although the effect of soaking RNAi was not observed, we decided to target the AK1 gene because there is already the report[1], and constructed the expression system of dsRNA in yeast.</p>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/83/Kyoto_fig3b.png" width="60%">
 
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<p class="caption"><b>Figure 3-b</b> Effects of soaking RNAi
 
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<i>B. xylophilus</i> were soaked into 2 mg/mL dsRNAs shown in Figure 3-a. After 4h incubation, nematodes were washed and incubated on M9 buffer plate (time=0h). Plates were examined for mortality of the nematodes up to 24h. The method of soaking RNAi was based on reference[1].
 
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</p>
 
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<h5 id="res4">4) Conduct feeding RNAi in yeast</h5>
 
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<p>In order to express AK1-dsRNA, we placed inverted repeat derived from AK1 ORF downstream of the Gal1 promoter of the part (<a href="http://parts.igem.org/wiki/index.php/Part:BBa_K517000">BBa_K517000</a>), and inserted a small loop sequence of 67-nt between repeats. There was a report that this loop sequence was effective when <I>S. cerevisiae</i> expressed long dsRNA[2]. Since <i>S. cerevisiae</i> has no Dicer homolog, dsRNA is not processed into siRNA. However, overexpression of dsRNA may be toxic to <I>S. cerevisiae</I>, so we adopted the Gal1 conditional promoter. When <I>S.cerevisiae</I> is cultured in the presence of glucose, this promoter is inactive, and many mRNAs are expressed when the carbon source of the medium is replaced with galactose. At the same time, we also used the GPD promoter (<a href="http://parts.igem.org/Part:BBa_K517001">BBa_K517001</a>) which is a constitutive expression type promoter. dsGFP with a sequence specific to GFP and was designed as a negative control. Outline of construction is shown below (Figure 4-a).</p>
 
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<br></br>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/2/28/%E3%82%A2%E3%82%BB%E3%83%83%E3%83%88_14%404x.png" width="70%">
 
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<p class="caption"><b>Figure 4-a</b> dsRNA expression vectors we used.<br>
 
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Left: Design of each dsRNA cassette. Plus strand and minus strand are tandemly transcribed as a single strand RNA.<br> To enhance dsRNA formation, a short hairpin loop sequence was inserted in between.<br>
 
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Right: Our plasmids used in this study. Gal1 promoter or GPD promoter was fused to dsAK1 or dsGFP (negative control) cassettes. The cassetes were cloned in YEPlac195 plasmid (2-micron, high-copy number plasmid).
 
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</p>
 
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<br>
 
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<p>We cultured plasmid-containing yeasts in several media, collected RNA, and quantified by qRT-PCR with the "loop" part as a target.Moreover, it is known that various viruses of dsRNA type exist in <I>S. cerevisiae</I>. As a factor closely related to the life cycle of such a virus, Ski gene group is known. Many of these are now revealing detailed functions. The Ski complex binds to the 3 'end of RNA and serves as a cofactor for RNA exosome, which is an exonuclease complex that degrades RNA in the 3-5 direction. By binding to the 3 'end to disband the higher-order structure of RNA, it makes the recognition of substrate by exosome efficient. Since the dsRNA virus is known to proliferate in the ski2Δ strain [3], it was hoped that the use of this strain would greatly increase the yield of the target dsRNA.
 
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</p>
 
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<p>
 
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The following is the result of qRT-PCR (Figure 4-c). First, expression of dsRNA was successfully detected when wild-type yeast into which Gal1 promoter-dsAK1 was introduced was induced by galactose. Almost the same values ​​are obtained even when the target of the primer set used for qRT-PCR is set to the loop portion or set within the AK1 gene. On the other hand, expression was suppressed as expected when replacing the medium with Glucose. From this, it was demonstrated that it is possible to conditionally induce long hairpin RNA expression using our plasmid.
 
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Interestingly, the expression in Ski2Δ strain is higher than that in WT strain (p <0.05). This indicates that in wild type yeast, Ski complex is degrading targeting foreign dsRNA in addition to RNA virus as expected. From these results, it was found that it is possible to raise the intracellular concentration of exogenous dsRNA by using yeast mutant strain.
 
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In GPD promoter (<a href="http://parts.igem.org/Part:BBa_K517001">BBa_K517001</a>), dsRNA could not be expressed. This part is composed of only the 112 bp sequence near the center out of the TDH 3 promoter (588 bp). Strong expression was confirmed when the 588 bp full-length promoter (<a href="http://parts.igem.org/Part:BBa_K530008">BBa_K 530008</a>) was used for eGFP expression experiments (Figure 2-a, 2-b), so we believe that there is a high probability that this part is defective.
 
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</p>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/1/12/Kyoto_fig4b.png" width="60%"></p>
 
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<p class="caption"><b>Figure 4-b</b> Quantification of dsRNA in <i>S. cerevisiae</i><br>
 
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Dr. Makoto Kitabatake (Institute for frontier life and medical science, Kyoto University) performed qRT-PCR using purified yeast total RNAs. The indicated strains were grown in SD-Glucose or SD-Galactose and harvested at the mid to late log phase. Total RNAs were isolated by MasterPure Yeast RNA purification kit (Lucigen) and analyzed by SuperScript III Platinum SYBR qRT-PCR kit (Thermo) after DNase treatment. The primers used in this assay are shown. Quantification of dsRNA was normalized by 25S rRNA. (n=3)</p>
 
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<br>
 
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<h5 id="res5">5) Observe that <I>B. xylophilus </I>feeds on yeast expressing dsRNA</h5>
 
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<p>We let <I>B. xylophilus</I> prey on the yeast prepared as described above and recorded the survival rate and behavior of nematodes as follows.</p>
 
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<p>We counted the number of surviving nematodes which fed on dsRNA / eGFP expressing yeast every other day. We also confirmed the survival rate among nematodes that showed fluorescence of eGFP.</p>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/9/9c/Kyoto_fig5abc.png" width="60%"></p>
 
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<p class="caption">
 
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<p class="caption"><b>Figure 5-a</b> Survival rate of <i>B. xylophilus</i> fed by yeast expressing dsAK1<br>
 
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Diploid yeast strain carrying the both dsAK1 and eGFP plasmids were grown in SD-Glucose (glu) or SD-Galactose (gal) medium and spread on new plates. ~100 nematodes were grown on each plate and examined by microscopy. (n=3) <br>
 
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<p class="caption"><b>Figure 5-b</b> Mortality of GFP(+) nematodes<br>
 
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Nematodes fed by diploid yeast carrying dsAK1 and eGFP plasmids were examied by fluorescence microscopy. Gal, yeast culture was prepared by SD-Galactose medium (dsRNA induction). Glu, the same yeast strain was grown in SD-Glucose medium (dsRNA repression). (n=3) <br>
 
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<p class="caption"><b>Figure 5-c</b> Survival rate of <i>B. xylophilus</i> fed by Ski2Δ strain<br>
 
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Feeding RNAi experiments were performed as in Figure 5-a. Ski2Δ strain instead of WT strain was used as prey. (n=1)
 
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</p>
 
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<br>
 
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<p>From the above results, the number of nematodes did not decline predominantly even when using yeast whose expression of dsAK1 was confirmed (Figure 5-a). According to previous experiments, when the nematodes cultured by eGFP(+) yeast, the rate of eGFP(+) labbeled nematodes was only about 30% (Figure 2-d). For this reason, even if dsRNA taken in kills nematodes, since the proportion of nematodes ingesting a sufficient number of yeasts to obtain the effect is not so high, there is a possibility that the effect given by dsRNA has been diluted. We focused only on nematodes that fed yeast and confirmed the mortality of fluorescent nematodes to evaluate the effect of dsRNA (Figure 5-b).
 
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Even in this case, we could not confirm the effect of dsRNA as expected.
 
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Moreover, the survival rate of nematodes is lower when using yeast cultured in +glu SD medium which should suppress the expression of dsRNA.
 
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The results were the same even when using ski2Δ yeast in which the expression level of dsRNA was increased (Figure 5-c).
 
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This seemingly contradictory result will be discussed later in the discussion.
 
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We thought that there might be some obstacle before dsRNA was taken up by nematodes and sought out the cause.</p>
 
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<h5 id="res6">6) Improve transport of mRNA to cytosol</h5>
 
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<p>As shown in the figure (Figure 6-a), the diameter of the stylet is very small, about a fraction of a single cell of yeast. For this reason, <I>B. xylophilus</I> seemed to draw out the cytoplasmic fraction, but large cellular componentns such as the nucleus may not be efficiently consumed by <I>B. xylophilus.</I></p>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/f/fd/Stylet.jpeg" width="60%"></p>
 
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<p class="caption">
 
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<b>Figure 6-a</b> Nematode’s stylet, diploid yeast, and haploid yeast. Scale bar : 10μm
 
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</p>
 
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<p>A number of studies have been done on the nuclear export of mRNA, and the basic mechanism has been elucidated. It is known that various RNAs such as mRNA, rRNA, tRNA, etc. are recognized by transporters specific to each type and pass through the nuclear pore complex[4]. However, since the dsRNA as prepared this time does not exist in nature, it is not known whether there is a transport factor that recognizes this RNA or whether it is efficiently transported out of nucleus.</p>
 
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<p>In order to prepare remedies for this problem, we tried experiments utilizing the REV factor of HIV-1 RNA, which is known to have the function of improving the efficiency of nuclear export of RNA.</p>
 
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<p>As shown in the figure, REV plays the role of carrying an unspliced RNA genome to the cytoplasm in the life cycle of HIV-1 (Figure 6-b). In the case of ordinary mRNA, there is a retention mechanism that prevent molecules retaining introns from transferring out of the nucleus, thus preventing the transport of immature mRNA. REV binds to a specific part (RRE: Rev responsive element) of the intron on the HIV-1 RNA genome and binds itself to the nuclear export factor CRM1, and overcomes such a retention mechanism and transports RNA to the cytoplasm[5]. Even if the dsRNA is not recognized as a nuclear export factor or even if it is retained in the nuclear retention factor, we thought that it is possible that the efficiency of nuclear export can be improved by inserting REV-RRE system, and provide these new parts to the iGEM community (<a href="http://parts.igem.org/Part:BBa_K2403000">BBa_K2403000</a>,
 
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<a href="http://parts.igem.org/Part:BBa_K2403002"> BBa_K2403002</a>)</p>
 
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<br></br>
 
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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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<p class="caption">
 
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<b>Figure 6-b</b> Rev protein induces nuclear export of RRE-containing RNAs.
 
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</p>
 
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<p>The results of microinjection of RI-labeled dsRNA with RRE into the nucleus of Xenopus oocytes are shown in the figure (Figure6c ~ Figure6f). Nuclear and cytoplasm were separated after a certain period of time following injection, RNA was recovered from each and analyzed . </p>
 
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<br></br>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/8/86/Kyoto_fig6c.png" width="50%"></p>
 
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<br></br>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/7/75/Kyoto_fig6d.png" width="60%"></p>
 
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<br></br>
 
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<p class="pic"><img src="https://static.igem.org/mediawiki/2017/6/6f/Kyoto_fig6fe.png" width="60%"></p>
 
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<br></br>
 
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<p class="caption">
 
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* Dr. Taniguchi, Institute for fronter life and medical science, Kyoto university, conducted an experiment on the process of treating RI on and after in vitro transcription.<br>
 
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<p class="caption"><b>Figure 6-c</b> Outline of Xenopus oocyte microinjection<br>
 
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<p class="caption"><b>Figure 6-d</b> RNAs produced by in vitro transcription<br>
 
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U6 and U6-RRE are used as controls.
 
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<br>
 
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<p class="caption"><b>Figure 6-e</b> Microinjection of dsGFP with or without RRE into Xenopus oocyte<br>
 
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Indicated RNAs with or without Rev protein were injected into Xenopus oocyte nucleus. The oocytes were dissected at the indicated time points (t=0, 60 min). The nuclear RNAs (N) and the cytoplasmic RNAs (C) were extracted and analyzed by PAGE. The left pannel shows dsGFP with RRE (GFP-RRE + GFPrev), the right pannel shows dsGFP without RRE (GFPfwd + GFPrev).<br>
 
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<p class="caption"><b>Figure 6-f</b> Long exposure
 
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</p>
 
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<p>
 
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As expected, when U6-RRE is injected together with buffer without Rev, U6-RRE remains in the nucleus, whereas U6 - RRE is injected with buffer containing Rev, U6-RRE was remarkably transported outside the nucleus. This result demonstrated that nuclear export of RNA is promoted depending on both the RRE sequence and the Rev protein in the case of U6 RNA originally staying in the nucleus. These effects indicate that these parts are promising as devices for efficiently transporting highly structured RNA, which is often used in synthetic biology, to the cytoplasm.(<a href=" http://parts.igem.org/Part:BBa_K2403000">BBa_K2403000</a>
 
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<a href="http://parts.igem.org/Part:BBa_K2403002">BBa_K2403002</a>)
 
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Unfortunately, transcription products of GFP-RRE were too thin to understand whether they responded to Rev. However, from the figure on the right of Figure 6-f, it was also found that dsRNA remained in the nucleus a lot. It is suggested that implementation of a system to promote nucleocytoplasmic transport is effective.
 
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Compared to the signal at T = 0, since many signals are lost at 60 minutes after injection, there may be a mechanism for degrading dsRNA in cells. In addition to promoting transportation efficiency, there will be room for improvement to improve stability.
 
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<br>
 
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<p>This is the Result obtained in this project.
 
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We would like to discuss Discussion on interpretation of Result and Future plan.</p>
 
 
   <h6>Reference</h6>
 
   <h6>Reference</h6>
 
       <ul class="reference">
 
       <ul class="reference">

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。

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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.