Part:BBa_K1486035

Yeast Optimized sfGFP-C + ADH1 terminator + CaUra3 Cassette

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 C-terminus (the last 24 amino acids) followed by the ADH1 terminator and a CaUra3 cassette containing a constitutive TEF promoter, a Ura3 gene from Candida albicans (allowing selection on media lacking uracyl) and a constitutive TEF terminator.

Use this part

To use this part and tag the protein Hog1, 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 Hog1 and the 50 first non-coding bases after the gene. To perform a protein complementation assay, we tagged the protein Pbs2 with another BioBrick: the BBa_K1486029.

Experience: Response to osmolar stress

The cells transformed with BBa_K1486029 and the 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).

Experience 2: response to acetic acid

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 plasmids we ordered to addgene:Plasmid 44873 pFA6a-link-yoSuperfolderGFP-CaURA3.

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 EcoRI site found at 779
  Illegal PstI site found at 468
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 779
  Illegal PstI site found at 468
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 779
  Illegal XhoI site found at 38
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 779
  Illegal PstI site found at 468
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 779
  Illegal PstI site found at 468
  
• 1000
  COMPATIBLE WITH RFC[1000]