One of the important goals of synthetic biology is to introduce new signalling pathways, such as CAR-T cancer immunotherapy. Due to the need for faster cellular responses to the input signals, synthetic biology is increasing its focus on regulating biological systems at the posttranslational level, such as proteolysis and phosphorylation. In the master's thesis, we constructed the concept of regulating the activity of the target protein through phosphorylation. For this purpose, we inserted into the firefly luciferase reporter protein the sequence for the SH2 binding domain and a substrate peptide that is recognised and phosphorylated by the corresponding kinase. The manipulation described above results in the target protein being inactive in its native state and its activity can be restored by phosphorylation with the appropriate kinase. To establish the most optimal relationship between the inactive and active state of firefly luciferase, we prepared a series of constructs with different linker lengths and different positions of inserted phospho-peptide binding domains and substrate peptides. We designed two one-molecular constructs; a split luciferase with an inserted phospho-peptide binding domain and a substrate peptide between N- and C-terminus and a split luciferase with an inserted substrate peptide between N- and C-terminus, and a phospho-peptide binding domain attached to the C-terminus with a flexible linker. We tested the concept with Lyn and Lck kinases, which are members of the Src family of non-receptor tyrosine kinases and are attractive therapeutic targets due to their role in pathological processes. In this thesis, by designing the phosphorylation-dependent activity of the target protein, we have laid the foundations for the design of a synthetic phosphorylation cascade in mammalian cells, which has two major advantages, namely (i) rapid response to environmental signals and (ii) reversibility. The system is designed in a modular way, i.e. it is scalable and allows the design of logic functions, phosphorylation cascades and positive and negative feedback loops.
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