Part:BBa_K1164004

pTRELX: Modified Gal1 promoter; gal4O replaced w/ tetO; repressable via lacO

This is a modified version of the native Gal1 promoter found in S.cerevisiae. The four gal4 binding domains present in the native promoter have been replaced by four tetO sites. This promoter may be activated by regulatory activators that are able to bind to tetO. In addition, a lacO operator has been introduced downstream of the TATA box, allowing for repression of gene expression by lacI.

The components of this part are sourced from the native S.cerevisiae genome and the Collins lab at Boston University. Overlap extension PCR was used to replace the gal4 binding domains in the native Gal1 promoter with tetO sites. The proximal region of the promoter, containing the lacO operators, was derived from the Collins lab.

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 465
  Illegal BamHI site found at 553
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 81
  
• 1000
  COMPATIBLE WITH RFC[1000]

The above data was collected from the following yeast strain:
ade2::Kanmx-rtact1-pgalLx-gfp
ade4::Natmx – rtact1-pgal-laci-bfp
In the above construct, pgallx drives the production of GFP and is repressed by Laci. As shown in the graph above, the repressor, Laci (tagged with BFP), remains relatively constant over the entire IPTG gradient: a chemical that represses the activity of Laci. Consequently, as IPTG concentration increases, Laci inhibition should be increasingly inhibited, resulting in more GFP being transcribed, which is supported by the above data. The levels of fluorescence then stabilize and achieve steady state.

The above data was collected from the following yeast strain:
ade2::Kan-rtACT1-pGal1-rtTA-mCherry-VP16
ade4::Nat-rtACT1-pTRE-LacI-BFP
In the above construct, PTRE (driving BFP) is activated by rtTA , which is driven by pGal and requires the presence of aTc. The levels of fluorescence then stabilize and achieve a steady level. The levels of fluorescence then stabilize and achieve a steady level. This graphic shows that as the aTc concentration increases, PTRE is being increasingly activated, resulting in greater BFP transcription. This is expressed as the increase of fluorescence in the graphic above.

Reference

1. Ellis T, Wang X, Collins JJ. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol. 2009; 27:46