Difference between revisions of "Part:BBa K3930015"

Latest revision as of 08:54, 17 October 2021

Constitutive promoter TEF1 Sequence and Features

Introduction

The promoter TEF1 is a constitutive promoter that comes from the plasmid p405TEF1 from Nicolas Buchler & Fred Cross (unpublished). The TEF1 promoter coming from the yeast Y. lipolitica is already described and used in part (BBa_K2117000), but ours comes from the S. cerevisiae genome.

Results

Production of β-ionone

All the experiments that characterized this part are related to the final construct pFRAMBOISE-notfused (BBa_K3930002) which was cloned into the S. cerevisiae LycoYeast strain. For more information on the experimental background, please refer to this part.

In the part (BBa_K3930002), the TEF1 promoter controls the expression of fyn-phCCD1, an enzyme that produce β-ionone from β-carotene. The production of β-ionone can thus be considered as a way to assess the functionality of the promoter TEF1. pFRAMBOISE-notfused was transformed into the S. cerevisiae LycoYeast strain. The β-ionone is very volatile and a common strategy to avoid losing these molecules during the culture is to grow the engineered microorganisms in a culture medium supplemented with an organic phase to trap the molecules of interest. The most common organic solvent used is dodecane for ionones (Chen et al., 2019; López et al., 2020). Figure 1 shows the GC-MS spectrum for the LycoYeast-pFRAMBOISE-notfused strain. A peak can be observed at the same retention time as the β-ionone standard for the induced LycoYeast-pFRAMBOISE-notfused strain. The mass spectra associated with this peak matched with the one obtained with the analytical standard. The β-ionone attribution was further confirmed by the NIST mass spectral library (National Institute of Standards and Technology). The production of β-ionone was successfully achieved with this construction. This means that the TEF1 promoter is functional to express gene in S. cerevisiae.

Figure 1: Production of β-ionone from LycoYeast-pFRAMBOISE-notfused

β-ionone is produced in vivo by our strain when it is induced by galactose. On the right are presented the mass spectra that correspond between the standard and the observed peak. First panel is the β-ionone standard. Second panel is the LycoYeast-pFRAMBOISE-notfused induced strain. Third panel is the LycoYeast-pFRAMBOISE-notfused non-induced strain. Forth panel is the LycoYeast WT strain.

We concluded the promoter TEF1 is functional under those lab conditions.