The design is divided into several parts:

1. A description of our basic tools regarding a particular problem
2. Constructing new parts
3. Cloning the constructs into yeast plasmid- pRS306 or pRS316 (and pSB1C3)
4. Transformation of the plasmids into yeast cells
5. Protein expression and puridication
6. Perform a inhibition test for Brettanomyces

Promoters:

ADH1: We chose the ADH1 promoter, as described on our parts page. On glucose, activity of the original ADH1 promoter decreases during late exponential, ethanol production growth phase (3). Because in the process of fermentation of the wine, the yeast uses the sugar and turns it into ethanol, and the levels of ethanol rises- this promoter is expressed and we control the time of our protein formation.

KIADH4:

Optimization for yeast:

Since we want to apply our own wine-making solutions, we plan to clone this genes into a plasmid and then for yeast, in order for the yeast to transcript it to protein.

The kp6 is encoded on specific medium-size (M) segments of the U. maydis viruse, and the miraculin gene from plant, the DNA sequence we found had to be optimized for yeast. We performed it using IDT tools.

α-factor secretion signal and histidine tag (6xHis-tag):

In order to examine and detect the expression of our desire protein, we chose to add these two elements to each of our constructs. The α-factor secretion signal is N-terminal secretion signal from S. cerevisiae alpha-factor. The alpha-factor was utilized to express the gene fusions in Saccharomyces cerevisiae and resulted in the efficient secretion of the foreign proteins into the culture medium (4)

Adding the 6xHis-tag designed for protein purification.Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Esch erichia coli and other prokaryotic expression systems. Bacterial cells are harvested via centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and enzymes such as lysozyme or any combination of these. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound divalent nickel or cobalt ions, which are available commercially in different varieties.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 SDS-PAGE and Western blotting.

ADH1 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, is 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. (5)

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

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.‏

1. Theerasilp, S., Hitotsuya, H., Nakajo, S., Nakaya, K., Nakamura, Y., & Kurihara, Y. (1989). Complete amino acid sequence and structure characterization of the taste-modifying protein, miraculin. Journal of Biological Chemistry, 264(12), 6655-6659.‏
2. Varela, C., Kutyna, D. R., Solomon, M. R., Black, C. A., Borneman, A., Henschke, P. A., ... & Chambers, P. J. (2012). Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts. Applied and environmental microbiology, 78(17), 6068-6077.
3. Ruohonen, L., Aalto, M. K., & Keränen, S. (1995). Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. Journal of biotechnology, 39(3), 193-203.‏
4. Bitter, G. A., Chen, K. K., Banks, A. R., & Lai, P. H. (1984). Secretion of foreign proteins from Saccharomyces cerevisiae directed by alpha-factor gene fusions. Proceedings of the National Academy of Sciences, 81(17), 5330-5334.‏
5. Curran, K. A., Karim, A. S., Gupta, A., & Alper, H. S. (2013). Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic engineering, 19, 88-97.‏
6. Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 60(5), 523-533.
7. Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429.‏