Difference between revisions of "Part:BBa K2117000"

Revision as of 10:52, 20 October 2020

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.

In order to bypass some of the limitations of the native TEF1 promoter, multiple tandem upstream activation sequences 1B (UAS1B) were fused with the lengthened or truncated TEF1 promoter. The expression of hrGFP under the hybrid promoters was measured using flow cytometry. The mean fluorescence levels of hrGFP were much higher when the reporter gene was expressed using hybrid TEF1 prompters fused with UAS1B tandem repeats than the native TEF1 promoter. This shows that expression levels of TEF1 promoter were enhanced by the UAS1Bs. In particular, TEF1 promoters fused with 16 tandem copies of UAS1B proved to have stronger expression compared to the TEF1 promoters fused with 8 tandem copies of UAS1B.

Impact of UAS1B8 and UAS1B16 on promoter strength decreases with an increase in the promoter length, indicting truncated version of TEF1 promoter coupled with UAS1B repeats results in highest levels of expression. This is most likely due to the fact that closer proximity of the UAS1B to the 5’ core promoter region allows for higher expression levels.

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