Team:Kyoto/Demonstrate

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Table of contents

• 1) Preparation of salt resistance enhancing plasmid in budding yeast
• 2) Preparation of yeast to incorporate Na+
• 3) Reduce the concentration of NaCl contained in the medium
• 4) Development of aggregation system

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 special care would be needed to create cells that absorb Na+ as a result of incorporating Na+, so that cells are not damaged. We searched the literature and found mangrin, a chaperone protein isolated from mangrove, cloned it and used it. As written on the link, Salinity tolerance was imparted by expressing mangrin in ENA1Δ strain sensitive to NaCl to take Na+. In addition, mild salinity tolerance was also observed with other plasmids, for example ZrGPD1 for producing compatible solutes glycerol and Avp1, AtNHXS1, SseNHX1 for transporting 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 NHA 1 and ENA 1,2,5, which drain Na+, knocked out by homologous recombination, and it was actually experimented that this strain stored Na+ in cells very efficiently we did (link). Furthermore, the expression of Zvp1, AtNHXS1 or SseNHX1 etc. showed that Na+ concentration in the cells increased.

3) Reduce the concentration of NaCl contained in the medium

A demonstration experiment was carried out to confirm how efficiently the Na+ added to the medium could be removed by optimizing the yeast strain by being assisted by modeling. As a result of the experiment, it was demonstrated that Na+ concentration remarkably decreased depending on time course after addition of yeast.

4) Development of aggregation system

Regarding SdrG, synthesis was only seen in the cell-free translation system, and detailed information such as whether the expression is successful in yeast or whether it is displayed on the cell surface or not could not be confirmed. However, regarding Fgβ, since yeast expressing it was immobilized by anti-Flag magnetic beads, it was evaluated that it was displayed on the surface of the cell as expected.