Difference between revisions of "Part:BBa K3629023"

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2-isopropylmalate synthase (LEU4) overexpression construct with Gibson homology

2-isopropylmalate synthase (LEU4) expression construct with Gibson homology, TEF intronic promoter (BBa_K3629001)double XPR2 terminator(BBa_K3629004),Yarrowia lipolytica LEU4 coding sequence (BBa_K3629019) and reverse gPCR primer binding site for Yarrowia lipolytica.

Usage and Biology

2-isopropylmalate synthase is the first enzyme involved in the biosynthesis of leucine. This is a very important enzyme in the pathway as it serves as a regulatory point in the biosynthesis pathway. The protein has a leucine binding domain which allows for down regulation of the pathway through negative-feedback inhibition of the enzyme. Expression of this enzyme especially on a strong promoter could be used to upregulate leucine synthesis and produce a leucine overproducing strain.

Expression of LEU4 is under the TEF intronic promoter which is the natural promoter found in the wild-type Yarrowia lipolytica for the Translation Elongation Factor 1 (TEF1) gene; it is a strong constitutive promoter. It also contains the first intron of the gene for stronger expression which helps with the overexpression of LEU4. XPR2 was used in the construct which is the terminator sequence from the Yarrowia lipolytica alkaline extracellular protease XRP2 gene.

Design

The construct is flanked with two Gibson homology sequences. Upon digestion with BbsI, the construct can be connected to the nourseothricin resistance expression construct on the 5' side and to either the mCherry or mCitrine expression construct on the 3' side. This allows for both a selection marker and a fluorescence protein to be easily added to the construct in one Gibson reaction.

An extra XPR2 terminator was included at the beginning of the construct, right before the promoter in case the construct is integrated into the coding sequence of a gene in the genome. This would stop the expression of that gene which might have otherwise interfered with the expression of LEU4 gene.

A reverse gPCR binding site was also included in the construct

Sequence and Features

References