The yeast two-hybrid system (Y2H) is an in vivo method for determining binary protein-protein interactions (PPIs). It is based on the modular transcriptional activator Gal4, which regulates genes involved in galactose metabolism. Gal4 binds to the upstream activating sequences of galactose promoters (UASGAL) and, in the presence of galactose, activates the transcription of genes. Gal4 consists of a DNA-binding domain (DBD) at the N-terminus, an activation domain (AD) at the C-terminus, and a homodimerization domain. The DBD and AD alone cannot interact and activate transcription. In Y2H, these domains combine only when an interaction occurs between the bait and the prey, bound to DBD and AD, respectively. The Y2H method is widely used and forms the basis for many other methods for determining PPIs in live yeast cells. Despite its advantages, the classical Y2H method has several limitations, including a high rate of false positives and negatives, it is time-consuming due to slow growth of the host strain, and it provides only qualitative PPI results without any knowledge about the affinity of PPI. The aim of this master's thesis was to develop a new host strain for the Y2H method to overcome the limitations of the classical Y2H.
The new host strain TZ88 is derived from the laboratory strain BY4741 and contains four reporter genes under the control of different galactose promoters: two reporter genes for auxotrophy complementation (HIS3 and URA3), a reporter gene that enables resistance to hygromycin B (HygR), and the reporter gene EGFP, whose expression is easily determined by measuring fluorescence intensity of its product, green fluorescent protein. All four reporter genes are expressed in the strain in the presence of both endogenous and exogenous Gal4 on glucose media. The TZ88 strain effectively distinguishes between negative control and known interaction partners on selection media without histidine, uracil, and in the presence of hygromycin B. Compared to the established strain PJ69-4a, the TZ88 strain is more selective as it does not require the addition of competitive inhibitor 3-AT to eliminate background expression of HIS3, but it is also less sensitive since the addition of 3-AT results in the loss of growth that relies on weaker interactors. The growth of TZ88 is better compared to PJ69-4a in the rich and complete synthetic medium, while growth in liquid selection media is comparable between the two strains. The EGFP reporter gene is expressed in both strains only in the presence of Gal4, but with bait and prey we were unable to detect its expression.
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