Part:BBa_K3927005

PGK1-TetO

Native PGK1p promoter with TetO operator downstream of TATA box such that promoter is repressed when TetO is expressed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Description

PGK1-tetO is a PGK1 S.cerevisiae promoter with a tet-operon inserted 20 basepairs downstream of it’s TATA box(Figure 1). TetR has been shown to bind to and repress tetO containing synthetic promoter in S.cerevisiae[1]. TetO was placed downstrem of the TATA box in order to construct a promoter that was constitutive unless TetO was expressed in the same cell

Design

PGK1-tetO construct schematic

Characterization

Figure 2: pTetRepress construct schematic tested in S.cerevisiae

Construct has been tested in an expression cassette containing PGK1-tetO upstream of a fluorescent mTurquoise gene, which simultaneously contained a tetR protein under the control of the blue light sensitive promoter C120-CYCp. In the presence of blue light and when transformed into a cell containing the appropriate factors to activate C120-CYCp, no difference was observed in the expression of mTurquoise(Figure 3) compared to the wildtype PGK1 promoter. This indicates that TetR did not successfully repress PGK1-TetO, and further engineering may be required to optimize this part.

Figure 3: pTetRepress as tested in S.cerevisiae containing transcription factors to activate C120-CYC promoter. No difference in expression was observed between cultures kept in the dark or light, indicating that the repression by TetR was not activated.

References

1. Pothoulakis, G., Ellis, T. Synthetic gene regulation for independent external induction of the Saccharomyces cerevisiae pseudohyphal growth phenotype. Commun Biol 1, 7 (2018). https://doi.org/10.1038/s42003-017-0008-0