Difference between revisions of "Team:Kyoto/Improve"
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| + | Figure1. Colony formation assay</p> | ||
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| + | Panel 1-3: NaCl 0 mM, Panel 4-6: NaCl 300 mM | ||
| + | Overnight cultures of the S. cerevisiae ena1- strain carrying each plasmid were serial diluted and spotted on a salt-containing SD plate. Colonies were photographed after 2 days incubation at 30oC. SseNHX1 and AtNHX1, separately cloned into pRS313, a low-copy-number plasmid, were expressed from TDH3 promoter. Control strain carrys empty vectors. | ||
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| + | As shown in Figure1, both SseNHX1 and AtNHXS1 show salt tolerance effect on ena1- strain when compared to the wild type. Remarkably, the effect was much greater in SseNHX1 strain than AtNHXS1 strain. | ||
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| + | <p>•Measurement of intracellular Na+ concentration by flame photometry</p> | ||
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| + | We evaluated the Na+ uptake efficiency by SseNHX1 and AtNHXS1 by measuring intracellular Na+ concentration. When SseNHX1-expressing strain was grown in 400 mM NaCl containing media, the average concentration of intracellular Na+ was 50.6 mM. On the other hand, when AtNHX1 was used, the intracellular Na+ was 49.2 mM. We concluded that SseNHX1 is slightly superior to AtNHXS1 in this assay. | ||
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Revision as of 18:35, 17 October 2018
タイトル？？
This year, we tried to improve AtNHXS1. AtNHX1 encodes a Na+/H+ antipoter located on the vacuole menbrane of A. thaliana. AtNHXS1 is a modified version of AtNHX1, created by DNA shuffling to improve NaCl resistance.
Based on a paper, we here created a new part, SseNHX1, which is an improved version of AtNHXS1. SseNHX1 was also created by DNA shuffling, with a combination of Salicomia europaea enzyme SeNHX1 and Suaeda salsa enzyme SsNHX1. It was reported that SseNHX1 show faster kinetics on the import of Na+ to the vacuoles than other relatives.
We characterized SseNHX1 by in vivo colony formation assay for the salt tolerance and flame photometry for the Na+ uptake into the cells.
Figure1. Colony formation assay
Panel 1-3: NaCl 0 mM, Panel 4-6: NaCl 300 mM Overnight cultures of the S. cerevisiae ena1- strain carrying each plasmid were serial diluted and spotted on a salt-containing SD plate. Colonies were photographed after 2 days incubation at 30oC. SseNHX1 and AtNHX1, separately cloned into pRS313, a low-copy-number plasmid, were expressed from TDH3 promoter. Control strain carrys empty vectors.
As shown in Figure1, both SseNHX1 and AtNHXS1 show salt tolerance effect on ena1- strain when compared to the wild type. Remarkably, the effect was much greater in SseNHX1 strain than AtNHXS1 strain.
•Measurement of intracellular Na+ concentration by flame photometry
We evaluated the Na+ uptake efficiency by SseNHX1 and AtNHXS1 by measuring intracellular Na+ concentration. When SseNHX1-expressing strain was grown in 400 mM NaCl containing media, the average concentration of intracellular Na+ was 50.6 mM. On the other hand, when AtNHX1 was used, the intracellular Na+ was 49.2 mM. We concluded that SseNHX1 is slightly superior to AtNHXS1 in this assay.
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Workshop with high school students
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Presentation at ISS
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Learning Lounge
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