Difference between revisions of "Team:Kyoto/Discussion"
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<h1>Discussion</h1> | <h1>Discussion</h1> | ||
| + | <ul class="discussion"> | ||
| + | <li><a href="#summary">1) The summary</a></li> | ||
| + | <li><a href="#method">2) The method of RNAi by feeding</a></li> | ||
| + | <li><a href="#dsRNA">3) Where is dsRNA in <i>S. cerevisiae</i>? Do <i>B. xylophilus</i> suck up dsRNA?</a></li> | ||
| + | <li><a href="#genes">5) Targeting essential nematode genes</a></li> | ||
| + | <li><a href="future">6) Future</a></li> | ||
| − | <h5>1) The summary</h5> | + | <h5><a name="summary">1) The summary</a></h5> |
<p>Pine-wilt disease, which is spreading all over the world, is one of the major plant diseases causing severe economic damage. A significant amount of money and labor are required for the treatment of forests each year. We focused on the cause of the disease, a nematode called <i>B. xylophilus</i>, and started our “B. x. Busters” project designed to create a new genetically modified microorganism exterminating <i>B. xylophilus</i>. We searched for a carrier of RNAi by feeding and revealed that under laboratory conditions, <i>B. xylophilus</i> preys on <i>S. cerevisiae</i> by sucking up the yeast’s insides using a straw-like stylus. We developed a reporter distinguishing <i>B. xylophilus</i> which ate <i>S. cerevisiae</i> by recording green fluorescence in the digestive track of <i>B. xylophilus</i> in live microscopy. In addition, we constructed a plasmid for <i>S. cerevisiae</i> expressing dsRNA, characterized its expression, and observed <i>B. xylophilus</i> which preyed on <i>S. cerevisiae</i> expressing dsRNA. These results, combined with our integrated Human Practices, could bring about promising new methodology in the fight against pine-wilt disease.</p> | <p>Pine-wilt disease, which is spreading all over the world, is one of the major plant diseases causing severe economic damage. A significant amount of money and labor are required for the treatment of forests each year. We focused on the cause of the disease, a nematode called <i>B. xylophilus</i>, and started our “B. x. Busters” project designed to create a new genetically modified microorganism exterminating <i>B. xylophilus</i>. We searched for a carrier of RNAi by feeding and revealed that under laboratory conditions, <i>B. xylophilus</i> preys on <i>S. cerevisiae</i> by sucking up the yeast’s insides using a straw-like stylus. We developed a reporter distinguishing <i>B. xylophilus</i> which ate <i>S. cerevisiae</i> by recording green fluorescence in the digestive track of <i>B. xylophilus</i> in live microscopy. In addition, we constructed a plasmid for <i>S. cerevisiae</i> expressing dsRNA, characterized its expression, and observed <i>B. xylophilus</i> which preyed on <i>S. cerevisiae</i> expressing dsRNA. These results, combined with our integrated Human Practices, could bring about promising new methodology in the fight against pine-wilt disease.</p> | ||
| − | <h5>2) The method of RNAi by feeding</h5> | + | <h5><a name="method">2) The method of RNAi by feeding</a></h5> |
<p>First, by feeding RNAi we aimed to knockdown the AK1 gene of <i>B. xylophilus</i>, since a previous study claimed success in targeting this gene with soaking RNAi [1]. However, we couldn’t reproduce any fatal effect to <i>B. xylophilus</i> just by soaking in RNA solution. Instead, we chose to feed <i>B. xylophilus</i> with <i>S. cerevisiae</i> expressing dsRNA by the conditional Gal1 promoter (BBa_K517001). We spread <i>S. cerevisiae</i> cultured in galactose medium onto no-nutrient agar medium containing no carbon source and fed it to <i>B. xylophilus</i>. This low nutrient condition prevents miscellaneous germs from proliferating, but at the same time the <i>S. cerevisiae</i> also lack nutrients. It probably led to autophagy. Under these conditions, it’s possible that low dosages of dsRNA were given to B. xylophilus</i> than we had initially anticipated. We recognize that we have plenty of room for improvement, optimizing the agar culture condition and preparing <i>S. cerevisiae</i> which maintaining dsRNA expression more stably for a longer period of time.</p> | <p>First, by feeding RNAi we aimed to knockdown the AK1 gene of <i>B. xylophilus</i>, since a previous study claimed success in targeting this gene with soaking RNAi [1]. However, we couldn’t reproduce any fatal effect to <i>B. xylophilus</i> just by soaking in RNA solution. Instead, we chose to feed <i>B. xylophilus</i> with <i>S. cerevisiae</i> expressing dsRNA by the conditional Gal1 promoter (BBa_K517001). We spread <i>S. cerevisiae</i> cultured in galactose medium onto no-nutrient agar medium containing no carbon source and fed it to <i>B. xylophilus</i>. This low nutrient condition prevents miscellaneous germs from proliferating, but at the same time the <i>S. cerevisiae</i> also lack nutrients. It probably led to autophagy. Under these conditions, it’s possible that low dosages of dsRNA were given to B. xylophilus</i> than we had initially anticipated. We recognize that we have plenty of room for improvement, optimizing the agar culture condition and preparing <i>S. cerevisiae</i> which maintaining dsRNA expression more stably for a longer period of time.</p> | ||
| − | <h5>3) Where is dsRNA in <i>S. cerevisiae</i>? Do <i>B. xylophilus</i> suck up dsRNA?</h5> | + | <h5><a name="dsRNA">3) Where is dsRNA in <i>S. cerevisiae</i>? Do <i>B. xylophilus</i> suck up dsRNA?</a></h5> |
<p> Our experiments confirmed SKI2Δ <i>S. cerevisiae</i> contains more dsRNA than wild type yeast. SKI complex is found in the cytoplasm. [2] In wild type yeast, SKI complex degrades dsRNA. Therefore, our results suggest that the dsRNA is partially in the cytoplasm. However, the results of Xenopus oocyte microinjection suggested that almost all the dsRNA is present in the nucleus. From our live imaging, it was not possible to see the yeast nucleus passed through the small diameter of the nematode’s stylus. If <i>B. xylophilus</i> doesn’t eat the yeast nucleus, the dsRNA will not reach the nematode’s body. Our further research confirmed that we could use the HIV Rev-RRE nuclear export pathway to help export dsRNA into the cytoplasm. Therefore, we predict that <i>S. cerevisiae</i> which has this function would improve dsRNA nuclear export to the cytoplasm, increasing the effective dose to predator <i>B. xylophilus</i>.<br> | <p> Our experiments confirmed SKI2Δ <i>S. cerevisiae</i> contains more dsRNA than wild type yeast. SKI complex is found in the cytoplasm. [2] In wild type yeast, SKI complex degrades dsRNA. Therefore, our results suggest that the dsRNA is partially in the cytoplasm. However, the results of Xenopus oocyte microinjection suggested that almost all the dsRNA is present in the nucleus. From our live imaging, it was not possible to see the yeast nucleus passed through the small diameter of the nematode’s stylus. If <i>B. xylophilus</i> doesn’t eat the yeast nucleus, the dsRNA will not reach the nematode’s body. Our further research confirmed that we could use the HIV Rev-RRE nuclear export pathway to help export dsRNA into the cytoplasm. Therefore, we predict that <i>S. cerevisiae</i> which has this function would improve dsRNA nuclear export to the cytoplasm, increasing the effective dose to predator <i>B. xylophilus</i>.<br> | ||
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| − | <h5>5) Targeting essential nematode genes</h5> | + | <h5><a name="genes">5) Targeting essential nematode genes</a></h5> |
<p>We tried to confirm whether RNAi by feeding has a fatal effect on <i>B. xylophilus</i> survival. However, we had no method to know the difference between life and death except for judging from the movement of <i>B. xylophilus</i> and, to make matters worse, dead <i>B. xylophilus</i> quickly dried-up and became difficult to detect. Therefore, it may be required to select a target gene (such as dpy) which expressed an obvious change in phenotype without killing, to establish the effect of RNAi by feeding in the future. (references)</p> | <p>We tried to confirm whether RNAi by feeding has a fatal effect on <i>B. xylophilus</i> survival. However, we had no method to know the difference between life and death except for judging from the movement of <i>B. xylophilus</i> and, to make matters worse, dead <i>B. xylophilus</i> quickly dried-up and became difficult to detect. Therefore, it may be required to select a target gene (such as dpy) which expressed an obvious change in phenotype without killing, to establish the effect of RNAi by feeding in the future. (references)</p> | ||
| − | <h5>6) Future</h5> | + | <h5><a name="future">6) Future</a></h5> |
<p>In the beginning, we intended to develop our “B. x. Busters” yeast as a biological pesticide, but we may need to solve many problems in advance, such as biosafety. As we described in our Human Practice (<a href=”https://2017.igem.org/Team:Kyoto/Human_Practices>https://2017.igem.org/Team:Kyoto/Human_Practices</a>), a promising strategy, attempting to create genetically-engineered pine trees expressing RNAi, is proposed. Our yeast system should be ideal for screening target genes and effective RNAi. RNAi by feeding with <p>S. cerevisiae</p> with optimization should reduce the time required for growing plants and would help us to realize our plan earlier. Accordingly, we plan to further improve our “B. x. Busters” and establish more effective RNAi by feeding. In addition to SKI2, we should verify the effect that other RNA metabolism-related factors may have on dsRNA accumulation. We can also use the Rev-RRE system to promote nuclear export, and improve our EGFP maker. When it comes to <i>E. coli</i>, many kinds of safety systems for using genetically-engineered <i>E. coli</i> in the environment have already been considered. Developing such systems for <i>S. cerevisiae</i> may bring about the possibility of their use not only in laboratories, but also in our environment.</p> | <p>In the beginning, we intended to develop our “B. x. Busters” yeast as a biological pesticide, but we may need to solve many problems in advance, such as biosafety. As we described in our Human Practice (<a href=”https://2017.igem.org/Team:Kyoto/Human_Practices>https://2017.igem.org/Team:Kyoto/Human_Practices</a>), a promising strategy, attempting to create genetically-engineered pine trees expressing RNAi, is proposed. Our yeast system should be ideal for screening target genes and effective RNAi. RNAi by feeding with <p>S. cerevisiae</p> with optimization should reduce the time required for growing plants and would help us to realize our plan earlier. Accordingly, we plan to further improve our “B. x. Busters” and establish more effective RNAi by feeding. In addition to SKI2, we should verify the effect that other RNA metabolism-related factors may have on dsRNA accumulation. We can also use the Rev-RRE system to promote nuclear export, and improve our EGFP maker. When it comes to <i>E. coli</i>, many kinds of safety systems for using genetically-engineered <i>E. coli</i> in the environment have already been considered. Developing such systems for <i>S. cerevisiae</i> may bring about the possibility of their use not only in laboratories, but also in our environment.</p> | ||
Revision as of 10:33, 1 November 2017
