Let’s design our project from the test tube to the wine bottle:

We chose the genes we want to express,our first step was designing a working expression cassette using different basic parts. Those parts may have regulatory functions like promoter and terminator sequences, or they may set the expressed protein’s location like the α-mating factor (α-MF) secretion signal. In addition, all the genes and parts which are not native to S.cerevisiae were optimized for yeast expression in order to gain maximal efficiency. We accomplished this with the help of IDT’s gene optimization tool.

The parts used are:

ADH1- Alcohol dehydrogenase promoter

We chose the ADH1 promoter, as described on our parts page. In a medium containing glucose, activity of the original ADH1 promoter decreases during late exponential growth phase, which is when ethanol is produced (1,2). We took the shortened version of the promoter which is 720 base pairs in length. Although the original part is inducible, the truncated version, which we used, is non-inducible to glucose and strongly expressed in yeast. This allows us to control the rate of protein synthesis and keep it constant, during changes in the glucose concentration in the medium.

α-mating factor (α-MF) secretion signal

The S.cerevisiae α-mating factor is widely used as a secretion peptide for recombinant proteins in multiple types of yeasts. The 86 amino acid peptide is attached to the N-terminal side of the target protein, and is translated as a pre-pro-peptide. Later, it is cleaved from the protein by the yeast’s endogenous enzymes in a 3 step process, before secretion (3,4).

Histidine amino acid tag (6xHis-tag)

Polyhistidine-tags are repetitive codon sequences coding for the amino acid Histidine. these tags are usually situated downstream from the gene of choice, and are utilized for the detection and purification of recombinant proteins. After lysation of the transformed cell, the target protein can be separated using Immobilized Metal Affinity Chromatography (IMAC). The 6XHis tag binds the protein to immobilized ions (usually Nickel) in the affinity column, and then the protein is eluted with concentrated Imidazole solution (5,8). Generally nickel-based resins have higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by Western blot.

ADH1- Alcohol dehydrogenase terminator

The importance of terminator choice has not been as widely studied as promoter activity. Usually only a few default terminators, such as those from the ADH1 gene,are used in yeast. The importance of 3′UTR regions as RNA stability elements has been well-established for bacterial systems. Efforts in prokaryotic systems have recently demonstrated that both terminators and designed 3′ UTR elements can fundamentally change heterologous expression level. (6)

After performing all of above, we had a pattern for our desired genes, for example:

Then, we sent them for synthesis in IDT.

Our second step was cloning the construct into the yeast plasmids which are:

• pRS306 - an yeast integrative plasmid which lacks an ORI (origin of replication) and has to be integrated into the yeasts chromosomes. This one as much smaller yield.
• pRS316 - a yeast centromere plasmid, it operates like a small independent chromosome but the yeast might get rid of it in next generations.
• pSB1C3 - a plasmid backbone for iGEM’s parts library.

The reason we selected to use 2 different yeast plasmid vectors, is to cover each other disadvantages.

After planning the genes, we started the laboratory work by inserting the gene into the desired plasmid.

At first we planned to do the RF-CLONING process, but after several failed attempts, we left this process and took a new approach.

Cloning the construct into yeast plasmid- pRS306 or pRS316

After planning the genes, we started the laboratory work by inserting the gene into the desired plasmid.

At first we planned to do the RF-CLONING process, but after several failed attempts, we left this process and took a new approach.

Plasmid Cloning by Restriction Enzyme Digest

Step 1: Digest The DNA

We add desire sites at the edge of our parts, that matches pRS306 / pRS316 / pSB1C3 - EcoR1 + Spe1, or EcoR1 + Pst1. Because there's some lose of DNA during the gel purification step, it is important to digest plenty of starting material. It is critical that as much of the recipient plasmid as possible be cut with both enzymes, and therefore it is important that the digest go at least 4 hours and as long as overnight.

Step 2: Ligate your insert into your vector:

Conduct a DNA Ligation to fuse the insert to the plasmid. We used two plasmids for yeast transformation, and one plasmid for submissing to iGEM HQ (pSB1C3).

The plasmids pRS306 and pRS316 are used for yeast expression, and they are some of a series of pBluescript-based integrating vectors differing in the yeast selectable marker gene. They also contain the URA3 domain - required for uracil biosynthesis. It’s yeast auxotrophic marker, that is required for later selection when transformed into URA- yeast.

Transform plasmid to yeast cells

The DNA used for transformation must carry a selectable marker whose presence can be detected by screening. Following a transformation, cells are plated on selective media that will allow transformed cells to grow. The plasmids that we are using carry a normal copy of the yeast URA3 gene, as well as the URA3 promoter, so the gene is regulated much like a normal chromosomal gene. Our yeast deletion strains were derived from strain BY920, which has the ura3∆0 allele. Complementation will occur because the plasmid carries a functional copy of the gene that is defective in the mutant host strain. The Ura3p protein produced from the plasmid-encoded URA3gene compensates for the ura3 deletion in the yeast chromosome, allowing transformed cells to grow in the absence of uracil, as shown below. Because of its reliability, many yeast transformation schemes rely on URA3 complementation to isolate transformants.

Protein expression in yeast, protein purification

after the yeast has matures it need to be transferred it to a liquid medium, where the secreted protein will be collected. the protein has a secretion signal and a histidine that binds to the nickel column.

Histidine-tagged proteins are commonly purified using Immobilized Metal Affinity Chromatography (IMAC). IMAC is based on the interaction between amino acid residues and divalent metal ions immobilized on resins. Histidine interacts most strongly, and histidine-tagged proteins have extra high affinity because of the multiple histidine residues.

The chemical imidazole is used to elute histidine-tagged proteins (6).

The IMAC relevant for our purpose is Nickel column.

The next step is using Western Blot technique in order to identify the desirable protein.

In this technique a mixture of proteins is separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labels antibodies specific to the protein of interest.The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible, then the protein is purified and eluted (7).

Perform an inhibition test for Brett

Santos, A., Navascués, E., Bravo, E., & Marquina, D. (2011). Ustilago maydis killer toxin as a new tool for the biocontrol of the wine spoilage yeast Brettanomyces bruxellensis. International journal of food microbiology, 145(1), 147-154.‏

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