Difference between revisions of "Part:BBa K2983074"

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RedStar2 expression cassette under the control of pTef1a (BBa_K2117000) promoter and Lip2 terminator

RedStar2 expression cassette under the control of pTef1 (BBa_K2117000) promoter and Lip2 terminator

Getting inspired by and improving the work of iGEM teams from previous years is a central aspect of the iGEM competition. The objective of our project is to produce medically-relevant Conjugated Linolenic Acids (CLnAs), which is a class of rare fatty acids with three conjugated bonds. In this project, we developed a launchpad for their bioproduction using the oleaginous yeast Yarrowia lipolytica, a powerful chassis organism, whose metabolism is naturally poised for lipid production. CLnAs are synthetized by bifunctional fatty acid conjugase / desaturase (FadX) enzymes from linoleic acid, a natural metabolite for our chassis. Thus, to convert it into a CLnA, only the presence of a FadX enzyme is necessary.

To drive the expression of FadX in Yarrowia lipolytica, the choice of the promoter is an important step: constitutive or inducible ? weak, medium or strong ? Several natural and synthetic promoters were characterized for Yarrowia lipolytica that allow tunable heterologous gene expression in this chassis (for a review see [1]). As producing a CLnA may lead to perturbations of vital cellular lipids (like the membrane glycerophospholipids) and thus reduce cell viability, we decided not to use an extremely strong promoter (like hp4d for instance [2]), nor a medium one (to put all odds on our side). We started our research from iGEM’s part registry and we quickly found in the database the pTef1 promoter (BBa_K2117000), a constitutive promoter native for the oleaginous yeast Y. lipolytica. It is a strong promoter that controls the expression of the translation elongation factor-1 alpha [3], a protein that is one of the most expressed in most cells (between 3-10% of the soluble proteins [4]). We will refer to BBa_K2117000 as pTef1a.

This promoter seemed suitable for our project, but had a major disadvantage: the presence of a BsaI site that makes it incompatible with the Type IIS RFC[10]-compatible Loop assembly system that we designed for Yarrowia lipolytica. To circumvent this incompatibility with the RFC[10] standard, we mutated the BsaI site (GGTCTC) to GGTCTg and thus created a new compatible part, BBa_K2983050, that we’ll refer to as pTef1b. A quick sequence analysis of BBa_K2117000 revealed several differences compared to wild-type pTef1 promoter (nucleotides 1227374 to 1226969) of Yarrowia lipolytica W29 chromosome C (GenBank Acc. n° CP028450.1). Three of the four mutations were introduced by the iGEM16_DTU-Denmark in order to remove two illegal restriction sites for BioBrick RFC[10]-compatibility (SpeI, PstI). As these sites are accepted in the Type IIS RFC[1000] standard, we created a closer to wild-type version of pTef1 promoter, BBa_K2983051, that has also a mutated BsaI site (GGTCTC to GGTCTg) which makes this part compatible with the iGEM Type IIS RFC[1000] standard. We will refer to BBa_K2983051 as pTef1c. Continuing our research, we discovered another version of the pTef1 promoter, which is shorter and Type IIS RFC[1000] compatible [5,6]: BBa_K2983052, that we’ll refer to as pTef1c.

Unpublished observations of our PI, Jean-Marc Nicaud, suggest that the presence of a 4 nucleotide sequence CACA just upstream the ATG of the gene may lead to increased gene expression. Thus, we added BBa_K2983053 to the list of pTef1 variants to test. We will refer to BBa_K2983051 as pTef1d.

A sequence comparison of all pTef1 variants is presented in figure 1. Apart from the above mentioned promoters, we included BBa_K2117000+scar, as this promoter was used to build BBa_K2117005 by standard 3A assembly which leaves TACTAG as a scar between the promoter and the ATG of the downstream gene.

Sequence and Features