Difference between revisions of "Team:Kyoto/Demonstrate"
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<h5 id="Preparation of salt resistance enhancing plasmid in budding yeast"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">1) Preparation of salt resistance enhancing plasmid in budding yeast </h5> | <h5 id="Preparation of salt resistance enhancing plasmid in budding yeast"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">1) Preparation of salt resistance enhancing plasmid in budding yeast </h5> | ||
| − | <p>We thought that | + | <p>We thought that in order to prepare yeast that absorbs Na +, special care that cells do not get damaged even if they take up Na + is required. We found mangrin, a chaperone protein isolated from mangroves by searching the literature, cloned it and used it. As written on the link, salt resistance was imparted to yeast of the strain by expressing mangrin in the ENA1Δ strain that is NaCl susceptible to incorporate Na +. Also, a gentle increase in salt tolerance was observed in the yeast with other plasmids, such as ZrGPD1 used to produce compatible solutes glycerol and Avp1, AtNHXS1, SseNHX1 used to transport Na + to vacuoles. As described above, in this study, we developed a parts collection that imparts salt tolerance to budding yeast by several different routes.</p> |
<h5 id="Preparation of yeast to incorporate Na+"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">2) Preparation of yeast to incorporate Na+</h5> | <h5 id="Preparation of yeast to incorporate Na+"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">2) Preparation of yeast to incorporate Na+</h5> | ||
| − | <p>We created a mutant yeast in which all of | + | <p>We created a mutant yeast in which all of NHA1 and ENA1,2,5 with a system to discharge Na + was knocked out by homologous recombination and showed that this strain actually stored Na + in cells very efficiently in the experiment. Furthermore, we showed that the expression of Zvp1, AtNHXS1 or SseNHX1 etc. increases Na + concentration in the cell. |
| + | </p> | ||
<h5 id="Reduce the concentration of NaCl contained in the medium"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">3) Reduce the concentration of NaCl contained in the medium</h5> | <h5 id="Reduce the concentration of NaCl contained in the medium"><img src="https://static.igem.org/mediawiki/2018/6/62/T--Kyoto--check.png">3) Reduce the concentration of NaCl contained in the medium</h5> | ||
| − | <p> | + | <p>We were assisted by modeling, optimized yeast strains, and carried out demonstration experiments to confirm how efficiently yeast can remove Na + actually added to the medium. As a result of the experiment, it was demonstrated that Na+ concentration remarkably decreased depending on time course after addition of the yeast.</p> |
<h5 id="Development of aggregation system (No check)"><img src="https://static.igem.org/mediawiki/2018/5/5b/T--Kyoto--null.png">4) Development of aggregation system</h5> | <h5 id="Development of aggregation system (No check)"><img src="https://static.igem.org/mediawiki/2018/5/5b/T--Kyoto--null.png">4) Development of aggregation system</h5> | ||
| − | <p>Regarding SdrG, synthesis was | + | <p>Regarding SdrG, we could only confirm that the synthesis was observed in the cell-free translation system, and we could not confirm the details, such as whether the expression is successful in yeast or whether it is displayed on the cell surface. However, regarding Fgβ, due to the fact that the yeast which expressed it was immobilized with anti-Flag magnetic beads, we evaluated that it was displayed on the surface of the cell as expected. |
</p> | </p> | ||
Revision as of 13:01, 26 November 2018
Our project Swallowmyces cerevisiae can be divided into four subtopics. For each individual topic, what is achieved is introduced here.
1) Preparation of salt resistance enhancing plasmid in budding yeast
We thought that in order to prepare yeast that absorbs Na +, special care that cells do not get damaged even if they take up Na + is required. We found mangrin, a chaperone protein isolated from mangroves by searching the literature, cloned it and used it. As written on the link, salt resistance was imparted to yeast of the strain by expressing mangrin in the ENA1Δ strain that is NaCl susceptible to incorporate Na +. Also, a gentle increase in salt tolerance was observed in the yeast with other plasmids, such as ZrGPD1 used to produce compatible solutes glycerol and Avp1, AtNHXS1, SseNHX1 used to transport Na + to vacuoles. As described above, in this study, we developed a parts collection that imparts salt tolerance to budding yeast by several different routes.
2) Preparation of yeast to incorporate Na+
We created a mutant yeast in which all of NHA1 and ENA1,2,5 with a system to discharge Na + was knocked out by homologous recombination and showed that this strain actually stored Na + in cells very efficiently in the experiment. Furthermore, we showed that the expression of Zvp1, AtNHXS1 or SseNHX1 etc. increases Na + concentration in the cell.
3) Reduce the concentration of NaCl contained in the medium
We were assisted by modeling, optimized yeast strains, and carried out demonstration experiments to confirm how efficiently yeast can remove Na + actually added to the medium. As a result of the experiment, it was demonstrated that Na+ concentration remarkably decreased depending on time course after addition of the yeast.
4) Development of aggregation system
Regarding SdrG, we could only confirm that the synthesis was observed in the cell-free translation system, and we could not confirm the details, such as whether the expression is successful in yeast or whether it is displayed on the cell surface. However, regarding Fgβ, due to the fact that the yeast which expressed it was immobilized with anti-Flag magnetic beads, we evaluated that it was displayed on the surface of the cell as expected.