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.
- Digest the DNA: We added restriction sites at the edge of our parts, that matches pRS306 / pRS316 / pSB1C3 - EcoR1 + Spe1, or EcoR1 + Pst1.
- Ligate insert into vector: Conduct a DNA Ligation in order to integrate the insert into the plasmid. The plasmids pRS306 and pRS316 contain the URA3 domain - required for uracil biosynthesis. It is a yeast auxotrophic marker, that is required for later selection when transformed into URA- yeast. The pRS306 contains two different ORI’s- one for replication in bacteria and the second for replication in yeast.
- Transformation of plasmids into yeast cells: The plasmid used for transformation must carry a selectable marker, the presence of which can be detected by selective screening. Following a transformation, cells are plated on selective media. The selective marker for our plasmids is a functioning copy of the yeast URA3 gene, as well as the URA3 promoter, so the gene is regulated much like a normal chromosomal gene. Our ura- yeast strains were derived from the strain BY920. If the yeast was successfully transformed, complementation will occur allowing the cells to grow in the absence of uracil. Because of its reliability, many yeast transformation schemes rely on URA3 complementation to isolate transformants.(7)
- After the yeast has matured it needs to be transferred to a liquid medium, where the secreted protein will be collected. The protein has a secretion signal and a histidine tag that binds to the nickel column.
- The next step is using the Western Blot technique in order to identify our target 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 (9).
- Perform an inhibition test for Brett: We had designed a theoretical test to check if our cloned yeast proved successful in inhibiting Brett. This test is comprised of two experiments with several treatments each. The first experiment was planned to be with a solid medium, the objective of using a solid medium is to monitor for the expansion of a halo. This medium will have contained Brett, which would be treated with a disc containing the following: 1) an extract of KP6 from our cloned yeast. 2) only the cloned yeast itself, to check if it succeeds in secreting the protein. 3) only un-cloned yeast. The second experiment would have involved a liquid medium, to mimic the anaerobic fermentation environment present in wine production, and the following treatments: 1) Brett with our cloned yeast. 2) Brett with SO2, for comparison with current methods employed by the industry. 3) Brett and KP6. As controls, we would use: 1) Brett on its own. 2) Brett with uncloned yeast. 3) only cloned yeast. Each treatment will be performed with different concentrations of the various constituents (Brett concentration remains constant).(10)
Now, when the molecular work is behind us it’s time for some old good winemaking procedure. We need to make sure that the Miraculin is indeed sweet just as the consumer wish for. We need to make sure that our toxin indeed eliminate Brett from the very beginning till the bottling step. In addition, a quality check of Resveratrol and ethanol concentrations is needed.
Finally, everything is designed, now it’s time to work full power! please see our results in the Notebook & Result page here.
- 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.
- Ruohonen et al. Modifications to the ADH1 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins. Journal of Biotechnology (1995) vol. 39 (3) pp. 193-203
- 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.
- Lin-Cereghino, G. P., Stark, C. M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., … Lin-Cereghino, J. (2013). The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris. Gene, 519(2), 311–317. http://doi.org/10.1016/j.gene.2013.01.062
- Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied microbiology and biotechnology, 60(5), 523-533.Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429
- 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.
- Xiao, W. (Ed.). (2006). Yeast protocols (pp. 33-p). Totowa: Humana Press, 114-116
- Paul N Hengen, Purification of His-Tag fusion proteins from Escherichia coli, In Trends in Biochemical Sciences, Volume 20, Issue 7, 1995, Pages 285-286, ISSN 0968-0004, https://doi.org/10.1016/S0968-0004(00)89045-3.
- Mahmood, T., & Yang, P. C. (2012). Western blot: technique, theory, and trouble shooting. North American journal of medical sciences, 4(9), 429.
- 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.