Difference between revisions of "Part:BBa K2665013"
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AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles and that original comes from Arabidopsis thaliana. | AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles and that original comes from Arabidopsis thaliana. | ||
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| − | <!-- Add more about the biology of this part here | + | We assembled '''[https://parts.igem.org/Part:BBa_K2225000 BBa_K2225000]''' with TDpromoter and CYC1 terminator to express the protein, and we added His-tag in case of Western blotting. |
| − | + | ||
| + | <!-- Add more about the biology of this part here--> | ||
| + | ==Usage and Biology== | ||
| + | This part sequence can be used directly because it contains promoter and terminator, but promoter is TDH3 promoter so it can't express in <i>E. coli</i>. AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles, so we used this part in order to increase the amount of Na+ uptake of yeast. | ||
| + | |||
| + | ==Characterization== | ||
| + | [[File:T--Kyoto--AtNHXS1.jpeg|400px]] <br> | ||
| + | [[File:T--Kyoto--cont.png|400px]]<br> | ||
| + | |||
| + | Pictures shown above is colonies of<i> S. cerevisiae</i> ΔENA1- strain on SD midium containing 400mM NaCl. In this spot assay, part BBa_K2665013 cloned to a yeast low-copy vector was used. <br> | ||
| + | This result shows that AtNHXS1 greatly contributes to salt tolerance of yeasts. | ||
| + | |||
| + | |||
| + | |||
| + | [[File:T--Kyoto--K_con.jpeg|400px]] <br> | ||
| + | [[File:T--Kyoto--Na con.jpeg|400px]]<br> | ||
| + | The graph shown above is K+ or Na+ concentration in cells of<i> S.cerevisiae</i> ΔENA1ΔNHA1 strain. Each gene in the graph was cloned to yeast vectors, which were introduced to yeasts. The word “high” means high copy plasmid and the word “low” means low copy plasmids. These transformed yeasts are cultured in SD containing 400mM NaCl. Detailed data are on our wiki.'''[http://2018.igem.org/Team:Kyoto Kyoto2018]'''<br> | ||
| + | These results show that AtNHXS1 contributes to accumulation of K+ and Na+ in a yeast cell.<br> | ||
| + | In addition, yeast harboring AtNHXS1 in high copy plasmids showed significantly low growth rate | ||
| + | |||
<!-- --> | <!-- --> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K2665013 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2665013 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | ===Reference=== | ||
| + | '''[https://parts.igem.org/Part:BBa_K2225000 BBa_K2225000]''' | ||
| + | |||
| + | <!-- --> | ||
| + | <span class='h3bb'>Sequence and Features</span> | ||
| + | <partinfo>BBa_K2403003 SequenceAndFeatures</partinfo> | ||
| + | |||
| + | |||
| + | <!-- --> | ||
Latest revision as of 23:00, 17 October 2018
TDH3-AtNHXS1-6xHis-CYC
AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles and that original comes from Arabidopsis thaliana.
We assembled BBa_K2225000 with TDpromoter and CYC1 terminator to express the protein, and we added His-tag in case of Western blotting.
Usage and Biology
This part sequence can be used directly because it contains promoter and terminator, but promoter is TDH3 promoter so it can't express in E. coli. AtNHXS1 is a Na+/H+ antiporter which is located in the membrane of vacuoles, so we used this part in order to increase the amount of Na+ uptake of yeast.
Characterization
Pictures shown above is colonies of S. cerevisiae ΔENA1- strain on SD midium containing 400mM NaCl. In this spot assay, part BBa_K2665013 cloned to a yeast low-copy vector was used.
This result shows that AtNHXS1 greatly contributes to salt tolerance of yeasts.
The graph shown above is K+ or Na+ concentration in cells of S.cerevisiae ΔENA1ΔNHA1 strain. Each gene in the graph was cloned to yeast vectors, which were introduced to yeasts. The word “high” means high copy plasmid and the word “low” means low copy plasmids. These transformed yeasts are cultured in SD containing 400mM NaCl. Detailed data are on our wiki.[http://2018.igem.org/Team:Kyoto Kyoto2018]
These results show that AtNHXS1 contributes to accumulation of K+ and Na+ in a yeast cell.
In addition, yeast harboring AtNHXS1 in high copy plasmids showed significantly low growth rate
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1446
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]