Team:NU Kazakhstan/Yeast



Yeast expression system

In this part of our project we were planning to construct the system that would express streptavidin on yeast surface, and biotinylated aptamers would be used to make sandwich biosensor for CEA detection. The plasmid that was planned to be modified was pYES2: we were planning to insert Aga2 gene followed by (G4S)3 linker and then streptavidin gene, which was present in 2013 distribution kit (biobrick part BBa_J36847). The expression of the construct would be under the control of GAL1 promoter. The yeast, where pYES2 plasmid containing the expression system was planned to be inserted, was Saccharomyces cerevisiae. S. cerevisiae is single cell, eukaryotic organism commonly used in biotechnology. It has several advantages, firstly, it is nonpathogenic, play a vital role in ethanol and baker’s yeast production, and then it is a eukaryotic organism and can be easily used in DNA recombinant technologies to express large proteins. The yeast itself has homothallic life cycle, i.e. it can exists as a haploid cell and after mating form a diploid cell. Diploid cells then can undergo meiosis and form spores. Mating types expresses Aga 1 protein on their surfaces which act like signaling protein. Another protein Aga 2 forms disulfide bonds with Aga 1 that is why it floats freely on the surface of the membrane. This Aga 1/Aga2 expression system can be used as a membrane directing mechanism, and different proteins can be expressed on yeast surface.

However, this part of the project was terminated because of the fact that we were not able to obtain mating type of yeast which was necessary for Aga2 gene, in association with Aga1 gene present in yeast itself, to express. Another obstacle was that we were not able to get the pure streptavidin sequence, thus gene construction was not performed.

This is how our gene construct would look like:

Gene sequence for our part would look like the following if it were put into standard plasmid:

We synthesized Aga2 gene in National Center for Biotechnology, Astana, however the whole biobrick part was not constructed because streptavidin part was not obtained.

In this project, we were also planning to use CdSe/ZnS QDs for conjugation to aptamers to create biosensor of sandwich manner. Two different aptamers against carcinoembryonic antigen (CEA) would be used. The first aptamer would become attached to streptavidin via biotin, and the second aptamer, which would be conjugated to QDs, would bind to the target protein itself. After that, fluorescent spectroscopy could be used to detect binding between conjugated aptamers and the corresponding target protein.

In order to see the specificity of binding between aptamers-QDs conjugates and the target, various imaging techniques such as fluorescence spectrophotometer, confocal laser microscopy, flow cytometry and other techniques can be applied.