Part:BBa_K2665016

TDH3-AtHKT1-6xHis-CYC

HKT family proteins are high K+ affinity transporters, and AtHKT1 is expressed in xylem of Arabidopsis thaliana. While HKT1 from wheat transport K+, AtHKT1 does not transport K+. Otherwise, AtHKT1 is better in Na+ transport. AtHKT1 inhibits the growth of S. cerevisiae in certain salt concentration.So, it probably transport Na+ into yeast cell.

Characterization

Pictures shown above is colonies of S. cerevisiae ΔENA1- strain on SD midium containing 400mM NaCl. In this spot assay, part BBa_K2665016 cloned to a yeast high-copy vector was used.
This result shows that AtHKT1 does not contribute to salt tolerance of yeasts. However, in our experiment, we could not conclude AtHKT1 increased salt-sensitivity because AtHKT1-expressing yeast showed remarkably slow growth in the medium without salt.

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 AtHKT1 contributes to accumulation of K+ and Na+ in a yeast cell.
This suggests that AtHKT1 is involved in afflux of NaCl and anchoring to yeast by a different mechanism from other localized pump expressed on vacuolar membrane.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1341
  Illegal BglII site found at 2598
  Illegal BamHI site found at 2394
  Illegal BamHI site found at 3030
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2690
  Illegal BsaI.rc site found at 2880
  

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2238
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1376
  

Reference

[4] A. Y. Ryss, O. A. Kulinich, and J. R. Sutherland, “Pine wilt disease: a short review of worldwide research,” For. Stud. China, vol. 13, no. 2, pp. 132–138, Jun. 2011.
[5] Y. Mamiya, “History of Pine Wilt Disease in Japan 1,” J. Nematol., vol. 20, no. 2, pp. 219–226, 1988.