Part:BBa_K1179014

VP16-Gal4 fusion protein which binds to UAS sites to be used for activation

This part encodes a transactivator protein that binds to the Gal4 galactose upstream activation sequence. The part is a fusion between the Gal4 DNA-binding domain and the viral VP16 transactivation domain. Gal4-VP16 has been proven to be a particularly powerful transactivator in mammalian cells, and is well suited to situations where levels of high transcriptional activation are necessary.

This part was further characterized by Harvard iGEM's 2019 team in fulfillment of their Bronze medal existing part characterization requirement. The authors of this characterization are Patrick Dickinson, Rahel Imru, and Teagan Stedman. This group worked under mentor Dr. Timothy Chang in Pamela Silver's lab at Harvard Medical School.

Gal4-VP16 can also be used as a component of the TANGO assay for measuring activation of G-protein coupled receptors. In the TANGO assay, a G-protein coupled receptor is modified to have a transcription factor (such as Gal4-VP16) attached to its C-terminus via a viral cleavage sequence. Under the right conditions, this viral cleavage sequence can be cleaved, resulting in the transcription factor being freed.

Here, we used Gal4-VP16 in such a context, with BFP (reporter) expression being driven by a 5xUAS reporter. We found that presence of the gal4-linked G-protein coupled receptor produced fluorescence which was all but invisible in control trials.

Trial 1: (g-protein coupled receptor-gal4 + cleavage protein + 5xUAS-BFP reporter)

Trial 2: (cleavage protein + 5xUAS-BFP reporter)

Trial 3: (untransfected HEK293T cells; note that coloring is different due to red, blue, and green channels being open for this photograph)

Comparing simple expression rates, we see 443 fluorescing cells in trial 1, with 2 fluorescing cells in trial 2. Trial 3 produces no fluorescing cells as that well was untransfected, yet it establishes that the observed change in fluorescence between trials 1 and 2 is likely not the result of autofluorescence.

Obviously, some variance in fluorescence could be explained by seeding density variation, but it is important to note that all three trials were seeded from the same, homogenous stock of HEK293T cells. Thus, a simple quantitative analysis allows us to arrive at the conclusion that the presence of gal4 is responsible for 5xUAS activation in greater than 99.5% of tested cells. This low level of leakage (<.5% of cells impacted) was to be expected, as the gal4-UAS system is orthogonal to human cells.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 461
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 380