Regulatory

Part:BBa_K3054001

Designed by: Dharmik Hitesh Patel   Group: iGEM19_Chalmers-Gothenburg   (2019-10-20)


pPGK1/pTEF1 dual promoter (yeast)

Dual promoter design

A dual promoter system generated by our team that consists of two strong promoters used in our lab from Saccharomyces cerevisiae. These promoters are placed in such a way that they can transcribe two protein sequences in opposite directions. Linker between these two promoters, 30bp of otherwise non-functional DNA, is not complementary to Saccharomyces cerevisiae genome. This helps in primer designing when extracting these promoters from Yeast genome and helps link 2 yeast promoters by fusion PCR. Diagram shows the design of dual promoter in general


Reference: Yasaman Dabirian, Xiaowei Li, Yun Chen, Florian David, Jens Nielsen, and Verena Siewers. Expanding the Dynamic Range of a Transcription Factor-Based Biosensor in Saccharomyces cerevisiae. ACS Synthetic Biology 2019 8 (9), 1968-1975. DOI: 10.1021/acssynbio.9b00144




Expression by the biobrick

Western Blot

Results of western blot of His-tagged proteins. From left to right the samples, together with their His-tagged genes are WT, WT, 1 (bphA1), ladder, 2 (bphA4), 3 (bphC), 4 (bphD), positive control

The biobrick has been verified by sequencing and the promoter was able to express the protein BphA1 in yeast. Only one clear image was obtained, verifying the function of the TEF1 promoter. With this image we can confirm that at least half of the promoter system 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 241


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