Difference between revisions of "Part:BBa K3927005"

Revision as of 13:59, 18 October 2021

PGK1-TetO

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

Sequence and Features

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

Usage

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.