1. Plasmid Selection Criteria

The plasmid vector type must be Saccharomyces cerevisiae since it is the only organism besides natural hosts (lavender and kiwi) that includes part of the MVA pathway.

The promoter must be constitutive in order for our solution to be scalable in the long run.

The plasmid must be high copy to obtain the respective yield of DNA to increase the volume of alcohol production.

The plasmid must contain a LEU2 promoter in order to be uptaken by genetically modified, leucine deficient yeast.

2. Our Plasmid and Expression System

The plasmid pJT1 (Addgene #98299) is designed for heterologous gene expression in Saccharomyces cerevisiae. Built on the pRS425 backbone, it supports stable replication and maintenance within yeast cells, while also containing a bacterial origin of replication and an ampicillin resistance marker for propagation in E. coli during cloning.

The plasmid expresses target genes under the control of strong, constitutive yeast promoters TEF1 and TEF2, which drive continuous transcription without the need for induction. This simplifies experimental workflows and ensures consistent gene expression under standard growth conditions.

Transcription in yeast is mediated by RNA polymerase II, with mRNAs undergoing typical eukaryotic processing steps such as capping, splicing, and polyadenylation. Each expression cassette includes appropriate yeast transcription terminators, such as TRPL3 or TRPL41B, to promote mRNA stability and efficient termination.

The expressed coding sequences are translated by the host’s ribosomes, producing functional proteins within the yeast cytoplasm. Because the promoters are constitutive and the plasmid maintains a relatively high copy number in yeast, pJT1 enables strong and reliable expression levels, making it well suited for metabolic pathway engineering and functional studies.

Selective pressure, typically via an auxotrophic marker encoded by the pRS425 backbone, ensures that only yeast cells retaining the plasmid continue to grow, maintaining stability of the construct across generations.