The design is divided into several parts:
- A description of our basic tools regarding a particular problem
- Constructing new parts
- Cloning the constructs into yeast plasmid- pRS306 or pRS316 (and pSB1C3)
- Transformation of the plasmids into yeast cells
- Protein expression and puridication
- 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:
