Part:BBa_K3927004

C120-CYC-TetR (S. cerevisiae)

This composite part encodes for part BBa_K3927001 C120-CYCp promoter controlling Part BBa_C0040, expressing TetR in the presence of blue light, followed by Part BBa_K1486025 ADH terminator. Dependent on EL222 expressed in situ., recommended that S. cerevisiae chassis has part BBa_K3570021 NLS-vp16-EL222 in order for this part to work.

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]

Design

Designs were remade for a third iteration of the blue light repression circuit (figure 1). Because directly using EL222 to hinder the promoter did not work, an inverter circuit was constructed such that repression of the promoter was reliant on EL222 driven expression. To achieve this, a TetO site was inserted into the identified repression site 25 base pairs downstream of the TATA box in the PGK1 promoter, and a secondary cassette containing the part BBa_C0040 coding for a tetR protein under the control of the initial C120-CYC promoter was conjugated to it. It was hypothesized that under blue light exposure, C120-CYC would activate the production of TetR, which would then repress the PGK1 promoter.

Figure 1:pTetrepress construct schematic.

Characterization

Overlap PCR was used to assemble the expression cassette C120-CYC-TetR-ADHt, and Gibson assembly was used to add this cassette alongside the TetO sequence into the blank repression plasmid, forming the plasmid pTetRepress. pTetRepress was transformed into the strain BY474B for testing.

Test: pTetRepress (BY474B) was cultured in darkness and blue light for 24 hours, and the fluorescence normalized to OD600.

Figure 1: pTetRepress(BY474B) cultured in darkness and blue light compared to the constitutive, unedited PGK1p promoter.

The third iteration of the secondary repressor module was still non-functional. Although it retained it’s baseline expression, exposure to light did not result in the expect reduction in expression. Further testing on the PGK1-tetO promoter, and the C120-CYC driven expression of tetR will be needed to fully troubleshoot this system. Further optimization work is required to troubleshoot the problem of this part.