Team:Kyoto/Improve

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Improving Parts

This year, we tried to improve AtNHXS1(BBa_K2225000). AtNHX1 encodes a Na+/H+ antipoter located on the vacuole menbrane of Arabidopsis thaliana. AtNHXS1 is a modified version of AtNHX1, created by DNA shuffling to improve NaCl resistance.[1]

Based on a paper, we here created a new part, SseNHX1(BBa_K2665005), 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.[2]

We characterized SseNHX1 by in vivo colony formation assay for the salt tolerance and flame photometry for the Na+ uptake into the cells.

•Colony formation assay

Figure1. Colony formation assay on SD medium.

Panel 1-3: NaCl 0 mM, Panel 4-6: NaCl 300 mM Overnight cultures of the S. cerevisiae ΔENA1 strain carrying each plasmid were serial diluted and spotted on a salt-containing SD plate. Colonies were photographed after 2 days incubation at 30℃. 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 ΔENA1 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 121.2 mM. On the other hand, when AtNHX1 was used, the intracellular Na+ was 89.7 mM. We concluded that SseNHX1 is superior to AtNHXS1 in this assay.

Figure2. Intracellular Na+ concentration (mM) of S. cerevisiae ΔENA1,2,5 strain expressing AtNHXS1 or SseNHX1 compared with wild type.

Based on the above two observations, we carefully compared the two homologous genes, SseNHX1 and AtNHXS1, and concluded that SseNHX1 is an improved version of AtNHXS1 in some features.

In order to calculate how much Na+ can be removed from the media, we mixed 1g yeast to 1 mL culture containing 100 mM NaCl. After incubation for 3.5h, aliquots were obtained and analyzed. The strain containing AVP1-SseNHX1 showed interesting result. With these two plasmids, the decrease of Na+ concentration in the media were fast. These results indicate that we have successfully demonstrate our devise, Swallowmyces cerevisiae, in a realistic condition.

Figure3. Sodium ion concentration of supernant of ΔENA1,2,5ΔNHA1 culture

References

[1]Xu, Kai Zhang, Hui Blumwald, Eduardo et al. (2010) A novel plant vacuolar Na+/H+antiporter gene evolved by DNA shuffling confers improved salt tolerance in yeast, Journal of Biological Chemistry Vol.285 Issue30 22999-23006 [2]G. Wu, G. Wang, J. Ji et al. (2015) A chimeric vacuolar Na+/H+antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast, Journal of Biotechnology Vol.203 1-8