Regulatory
TEF1p

Part:BBa_K2117000

Designed by: Isabella Loft   Group: iGEM16_DTU-Denmark   (2016-10-14)
Revision as of 05:15, 25 October 2020 by ArshiaMostoufi (Talk | contribs)


Constitutive TEF1 promoter native to Yarrowia lipolytica

Usage and Biology

The translation elongation factor-1α (TEF1) promoter is a strong constitutive promoter. Native for the oleaginous yeast Yarrowia lipolytica.

This promoter is often used for isolation of enzyme genes by expression cloning. However, the promoter is usually not recommended for heterologous production due to early expression of heterologous genes can be detrimental for cell growth.


Improvement by Evry_Paris-Saclay 2019

This part is functional as a promoter in the oleaginous yeast Yarrowia lipolytica, but it is not compatible with the Type IIS RFC[1000] standard and thus cannot be used in cloning experiments using the Golden Gate technique. As this is a powerful molecular biology technique that allows scarless assembly of a large number of DNA fragments and now it is fully supported by iGEM, we improved this part to make it compatible with the Type IIS RFC[1000] standard.

Thus, we have successfully built 4 versions of the pTef1 promoter: pTef1c (BBa_K2983050), pTef1d (BBa_K2983051), pTef1e (BBa_K2983052) and pTef1f (BBa_K2983053) which are able to drive the expression of a reporter gene in the oleaginous yeast Yarrowia lipolytica at an equivalent strength as this part already present in the iGEM Registry, pTef1a (BBa_K2117000).

Full details are available on the improved pTef1 promoter variants Part's Main Pages: BBa_K2983050, BBa_K2983051, BBa_K2983052 and BBa_K2983053.

Characterization from iGEM20_Calgary

Expression of the reporter gene, hrGFP, under several endogenous Yarrowia lipolytica promoters were tested using flow cytometry. The promoters used in this study include TEF1, EXP, FBA, GPAT, GPD, YAT, and XPR2. Out of the endogenous promoters tested, EXP and TEF1 showed the highest levels of expression for hrGFP, measured as mean fluorescence using a flow cytometer. However, expression levels of these two strong native promoters might sill be too low for some engineering purposes such as metabolic engineering. Therefore, a hybrid TEF1 promoter could be constructed to help improve the expression level of this native promoter.

Another study by Hong et al. compared the expression levels of TEF1 promoter to other native Y. lipolytica promoters such as FBA1, TDH1, and GMP1 using a quantitative fluorometric assay. The FBA1 promoter was shown to have the highest expression levels followed by the TDH1 promoter. GPM1 and TEF1 had similar strengths and were the weakest of the native promoters tested. In particular, the FBA1 promoter had 3.3 times higher transcriptional activity than the TEF1 promoter, when the mRNA abundance from these promoters were measured.

References

Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11

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.rc site found at 2


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