Difference between revisions of "Team:Kyoto/Design"
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<p class="honbun">We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(<i>Bruguiera sexangula</i>), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]<br>ZrGPD1 and ZrFPS1 is derived from <i>Zygosaccharomyces rouxii</i>. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。 | <p class="honbun">We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(<i>Bruguiera sexangula</i>), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]<br>ZrGPD1 and ZrFPS1 is derived from <i>Zygosaccharomyces rouxii</i>. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。 | ||
</p> | </p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2018/2/22/T--Kyoto--kyapucha.png" width="30%"> | ||
<center><img src="https://static.igem.org/mediawiki/2018/7/77/T--Kyoto--Designfig1.png" width="30%"><p>Figure1:酵母の耐塩性に貢献する３つのタンパク質を表した図。(聞くならmangrin→續さん、Zr:島添君)</center> | <center><img src="https://static.igem.org/mediawiki/2018/7/77/T--Kyoto--Designfig1.png" width="30%"><p>Figure1:酵母の耐塩性に貢献する３つのタンパク質を表した図。(聞くならmangrin→續さん、Zr:島添君)</center> | ||
Revision as of 20:44, 16 October 2018
Introduction
私たちは、Na+を効率よくとりこむusefulな酵母を作成し、モデリングによりある量のNa+を回収するのに必要な酵母投入量を割り出すことを目指しました。そのために、たくさんの研究がなされているシロイヌナズナや、塩生植物の遺伝子をもとにして、効率よくNa+を取り込むような系をデザインしました。
そしてバイオセーフティーのために酵母を凝集させることを試みました。
1)Halotorelance of Yeast
We tried to enhance halotolerance of yeast in order to make biological devices work even under high salt concentration. For this, we focused on 3 genes, Mangrin, ZrGPD1 and ZrFPS1. Mangrin is derived from a halophyte plant, Mangrove(Bruguiera sexangula), and encodes a shaperon like protein which is already confirmed to express in yeast. It helps proteins exist stably under high salt concentration. A paper has showed that only 71 amino acids of all the sequence is requied for the function, so we used the functional domain.[1]
ZrGPD1 and ZrFPS1 is derived from Zygosaccharomyces rouxii. In Japan, it's very popular because used for create soy source. ZrGPD1 encodes the glycerol-3-phosphate dehydrogenase(参考) and related to glycerol synthesis. ZrFPS1 encodes a putative glycerol transporter and inhibit its efflux. Glycerol works as a conpatible solute, so they are expected to work for increase the osmotic torelance and salt one.[2] By introducing these proteins, We tried to expand the range yeast can be addapted. その導入により酵母の適応塩濃度範囲をexpandします。
Figure1:酵母の耐塩性に貢献する３つのタンパク質を表した図。(聞くならmangrin→續さん、Zr:島添君)
塩吸収酵母
私たちは、酵母の細胞膜上、そして液胞膜上のを遺伝子工学的に改変することによりNa+取り込み系の実現を試みました。次のセクションから詳しく記述します。
Modification of Transporters on Cellular Membrane
細胞膜上のNa+輸送に関わるトランスポーターをノックアウトしたり導入したりすることで、細胞膜のNa+透過性を上げ、速度論的にNa+取り込みをimproveします。
・Na+を外部に流出するトランスポーターとして、ENA1,NHA1に注目し、以下のノックアウト株作成を試みました。
NHA1Δ、ENA1Δ、NHA1ΔENA1Δ、ENA1,2,5Δ、ENA1,2,5ΔNHA1Δ(正しい表記がわかりません)
ENA1 is nearly identical P-Type ATPases localized oncellular membrane and the first member of a tandem array of genes, and proved [3]
外のトランスポーターを×しているイラスト(Aachenを参考に。マングリンたちは灰色などにして目立たなくする)ENA1: the first member of a tandem array of genes encoding nearly, but not perfectly, identical P-Type ATPases.[3]()
NHA1:
・Na+を内部に取り込むトランスポーターとして、シロイヌナズナ由来のAtHKT1,アイスプラント由来のMcHKT2に注目しました。どちらのパフォーマンスがいいか選別します。いい方を導入し、Na+の取り込み促進を狙います。
外のトランスポーターは灰色とかで目立たなくして、細胞膜上でNa+を取り込むイラスト。AtHKT1:木部に発現するタンパク質で、Na+の輸送に関わるMcHK2:AthKT1のホモログで、塩耐性の強いアイスプラント由来のもの(担当、童と仲里さん)
(McHKT2はコンストできなかった、と正直にリザルトで書こうと思う)
Modification of Transporters on Vacuolar Membrane
Na+は様々な酵素の活性を阻害するので(参考)、液胞に隔離させるために、AntiporterNHX1とH+-PpaseAVP1を導入することでNa+取り込み機構を構築します。 NHX1として、シロイヌナズナ由来のAtNHX1をDNAシャッフリングにより活性を高めたAtNHXS1と、2種類の塩生植物のNHX1をDNAシャッフリングしたSseNHX1の２つがあり、どちらがよりよいパフォーマンスをするか選別します。 液胞膜上にトランスポーターがあってNa+を取り込むイラスト AtNHXS1: SseNHX1:(担当田向君だったが)
凝集酵母
私たちの酵母によって目的の塩濃度まで下げたあと、酵母を回収しやすくするためにそれらを凝集させる系の確立を目指しました。そのためにsurface displayを介してSdrG-FgβF3結合という共有結合に匹敵するほど強力なタンパク質間結合を利用しました。 surface displayにおいて、パッセンジャーを表層提示するためにsed1 anchoringドメインを用いました。
表層提示してくっついてるイラストReference
- [1] R. Haro, B. Garciadeblas, A. Rodriguez-Navarro (1991) A novel P-type ATPase from yeast involved in sodium transport, FEBS Letters Vol.291 Issue2 189-191
- [2] Jos6 A. Miirquez, Ramdn Serrano (1996) Multiple transduction pathways regulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast, FEBS Letters Vol.382 Issue1-2 89-92
- [1] A. Yamada, T. Saitoh, T. Mimura et al. (2002) Expression of Mangrove Allene Oxide Cyclase Enhances Salt Tolerance in Escherichia coli, Yeast, and Tobacco Cells, Plant and cell physiology 903-910
- [2] Hou,Lihua Wang,Meng Wang,Cong Wang,Chunling Wang,Haiyong (2013) Analysis of salt-tolerance genes in zygosaccharomyces rouxii, Applied Biochemistry and Biotechnoloogy 1417-1425 [4] A. Y. Ryss, O. A. Kulinich, and J. R. Sutherland, “Pine wilt disease: a short review of worldwide research,” For. Stud. China, vol. 13, no. 2, pp. 132–138, Jun. 2011.
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- [8] Kyoto Association for the Promotion of Traditional Culture of forest, “Danger of Kyoto's three representative mountains,” 2007. [Online]. Available: http://www.kyoto-dentoubunkanomori.jp/topics/img/brochure.pdf. [Accessed: 21-Oct-2017].
- [9] T. Kiyohara and Y. Tokushige, “Inoculation Experiments of a Nematode, Bursaphelenchus sp., onto Pine Trees,” J. JAPANESE For. Soc., 1971.
- [10]C. Vicente, M. Espada, P. Vieira, and M. Mota, “Pine Wilt Disease: a threat to European forestry,” Eur J Plant Pathol, vol. 133, pp. 89–99, 2012.
- [11]Rejendra Singh and Swastik Phulera, “Plant Parasitic Nematodes: The Hidden Enemies of Farmers,” Reserch gate, 2015.
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- [13]A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello, “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, vol. 391, no. 6669, pp. 806–811, Feb. 1998.
