Regulatory

Part:BBa_K3927005

Designed by: Chew Chin Wei   Group: iGEM21_NUS_Singapore   (2021-10-13)
Revision as of 14:01, 18 October 2021 by CWChew (Talk | contribs) (Usage)


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.


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