Part:BBa_K1486029

Yeast Optimized sfGFP-N + ADH1 terminator + Kanamycin resistance

This part is a protein tag to be used with the genomic integration technique (homologous recombination) on the budding yeast S.cerevisiae. It consists of a 13 a.a long linker, the yeast optimized sfGFP N-terminus (the first 214 amino acids) followed by the ADH1 terminator and a KanMX cassette containing a constitutive TEF promoter, a Kanamycin resistance (works against geneticin) and a constitutive TEF terminator.

Use this part

To use this part and tag the protein Pbs2, we attached 50 bases flanking regions to both 5' and 3' ends via PCR. Flanking regions are DNA sequences homologous to the yeast genome, targeting the genomic site of insertion. In our case, they were the last 50 coding bases of the protein Pbs2 and the 50 first non-coding bases after the gene. To perform a protein complementation assay, we tagged the protein Hog1 with another BioBrick : the BBa_K1486035.

Experience 1: Response to osmolar stress

The cells transformed with BBa_K1486029 and BBa_K1486035 were stressed with different concentrations of glucose and NaCl, while we read the fluorescence around 510nm. We distributed 135ul of cell culture (centrifugated and resuspended in PBS buffer to an OD of ~400), to which we added 15ul of 40% glucose (Gluc A), 30% (Gluc B), 20% (Gluc C), NaCl 1.4M (NaCl A), NaCl 1M (NaCl B) and NaCl 0.5M (NaCl C). Visit our wiki for more informations on the protocol.

Experience 2: response to osmolar stress

Cells transformed with BBa_K1486029 and the BBa_K1486035 were imaged by fluorescence microscopy before and after stress with acetic acid 3.6% (final concentration).

Experience 3: response to acetic acid (bis)

As above, the cells were stressed with acetic acid, and the fluorescence at 510nm was analysed over time.

Source

It was extracted by PCR from the plasmid we ordered to addgene:44901 pFA6a-link-yoSuperfolderGFP-Kan.

Reference

Improved Blue, Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae. Lee et al (PLoS One. 2013 Jul 2;8(7):e67902. doi: 10.1371/journal.pone.0067902. Print 2013. PubMed)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1035
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1035
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1035
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1035
  
• 1000
  COMPATIBLE WITH RFC[1000]