Projects / Programmes
Design of a fast signaling and logic processing pathway based on orthogonal proteases
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| P004 |
Natural sciences and mathematics |
Biochemistry, Metabolism |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
synthetic biology, coiled-coils, protein signaling, site specific protease, split protein
Researchers (14)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
14360 |
PhD Mojca Benčina |
Biotechnology |
Researcher |
2018 - 2021 |
392 |
| 2. |
35277 |
PhD Tina Fink |
Biochemistry and molecular biology |
Researcher |
2018 - 2020 |
25 |
| 3. |
17915 |
PhD Helena Gradišar |
Biotechnology |
Researcher |
2018 - 2020 |
130 |
| 4. |
38164 |
Van Thai Ha |
Pharmacy |
Researcher |
2020 |
12 |
| 5. |
28881 |
PhD Karolina Ivičak Kocjan |
Biotechnology |
Researcher |
2018 - 2020 |
54 |
| 6. |
06628 |
PhD Roman Jerala |
Biochemistry and molecular biology |
Head |
2018 - 2021 |
1,190 |
| 7. |
34252 |
Tina Lebar |
Biochemistry and molecular biology |
Researcher |
2018 |
67 |
| 8. |
52428 |
Sanjin Lulić |
|
Technical associate |
2019 - 2020 |
0 |
| 9. |
17917 |
PhD Andreja Majerle |
Biotechnology |
Researcher |
2018 - 2021 |
92 |
| 10. |
53353 |
Klemen Mezgec |
Biochemistry and molecular biology |
Researcher |
2019 - 2021 |
3 |
| 11. |
23939 |
PhD Martina Mohorčič |
Biotechnology |
Researcher |
2018 - 2021 |
30 |
| 12. |
38275 |
Anja Perčič |
|
Technical associate |
2020 - 2021 |
0 |
| 13. |
50616 |
Arne Praznik |
Biochemistry and molecular biology |
Researcher |
2018 - 2021 |
35 |
| 14. |
55061 |
Tina Strmljan |
|
Technical associate |
2021 |
0 |
Organisations (2)
Abstract
Cells are constantly exposed to versatile signals to which they have to respond accordingly. Signal transduction is an important biological process that cells use to detect, recognize and process versatile chemical or physical signals from the environment of from endogenous processes. In addition to the existing signaling and processing circuits we can endow cells with new processing and sensing logic, which has been used for innovative therapies such as e.g. in CAR T cell cancer immunotherapy or in optogenetics. So far engineering of cellular logic circuits that process the selected signals has been done at the level of transcriptional regulation. We and others have demonstrated that reliable and robust logic circuits can be introduced into different cell types by the use of orthogonal designed transcriptional regulators. A drawback of the transcriptional signaling pathway is however that it has relatively slow response time, on the order of hours to days, as it requires transcription and translation of new proteins and/or modification of the chromatin. For faster response to signals cells use natural protein-based signal transduction pathways which can respond in second to minutes as the synthesis of new proteins is not required. This is particularly important for the physiologically relevant processes which require fast response, within minutes, such as e.g. response to the increased concentration of a glucose, which results in the secretion of insulin. Those signaling pathways are based on protein modification and/or association and are more demanding to engineer as they are based on specific protein-protein interactions.
Here we propose to design a bottom-up fast protein-based signaling and logic processing pathway, which will be capable to integrate several signals into an appropriate cellular response. Designed signaling pathway will be based on the combination of split orthogonal proteases and designed coiled-coil based interactions and its function will be demonstrated in mammalian cells for potential therapeutic applications.
This strategic goal of the project will be implemented by the following aims that will design, test and demonstrate the function of the designed proteolysis and coiled-coil interaction-based signaling and logic system (PCCS) based on the following Workpackages: WP1, Design a set of split orthogonal proteases and test their function in mammalian cells; WP2, Design of intramolecular antiparallel coiled-coil sequences that can be displaced by a strong binding CC peptide upon proteolytic cleavage at engineered sites; WP3: Implementation of the PCCS-based signaling pathways and logic functions in mammalian cells and WP4: In vivo implementation and validation of designed fast response signaling pathway (PCCS).
Project will be executed by a team of experts in protein engineering and mammalian cell synthetic biology using a range of methods from molecular and mathematical modeling of biological processes, biophysical characterization, mammalian cell biology, fluorescence microscopy, flow cytometry to in vivo experiments.
The expected result of the project will represent the first case of a designed signaling pathway in mammalian cells. Previously attempts have been made to transfer components of the signaling pathways from yeast to mammalian cells, however completely de novo signaling pathway has not been designed before. There are many cases where a fast response of cells to the external or internal signal is required. Project will also result in the design of the set (toolbox) of split orthogonal proteases as well as the set of coiled-coil based switches that will be useful for other researchers. The proposed system therefore represents an important advance for synthetic and molecular biology and could have many fundamental and technological applications.
Significance for science
The expected results will represent the first case of a completely bottom up designed signaling pathway in mammalian cells absed on proteolysis. Project will result in the design of the large set (toolbox) of functional split specific proteases that will be useful for other researchers, as so far the biotechnological application has been almost exclusively limited to the TEVp. There are many cases where a fast response to the signals is required, such as for example the response of the organism to the glucose levels via insulin secretion, which might become feasible based on the completely designed cellular circuit strategy. The proposed system therefore represents an important advance for synthetic biology and could have many biotechnological applications.
Project will be performed by a project group that has an excellent track record in synthetic biology, protein biochemistry, biotechnology and mammalian cell biology. Project group comprises experts in molecular modelling, synthetic biology and mammalian cell biology. Project groups has available all equipment and technical expertise to achieve goals of the project. PI is also active in several networks in synthetic biology at the European level and results of this project can be disseminated to a wider research community.
Significance for the country
The expected results will represent the first case of a completely bottom up designed signaling pathway in mammalian cells absed on proteolysis. Project will result in the design of the large set (toolbox) of functional split specific proteases that will be useful for other researchers, as so far the biotechnological application has been almost exclusively limited to the TEVp. There are many cases where a fast response to the signals is required, such as for example the response of the organism to the glucose levels via insulin secretion, which might become feasible based on the completely designed cellular circuit strategy. The proposed system therefore represents an important advance for synthetic biology and could have many biotechnological applications.
Project will be performed by a project group that has an excellent track record in synthetic biology, protein biochemistry, biotechnology and mammalian cell biology. Project group comprises experts in molecular modelling, synthetic biology and mammalian cell biology. Project groups has available all equipment and technical expertise to achieve goals of the project. PI is also active in several networks in synthetic biology at the European level and results of this project can be disseminated to a wider research community.
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
Interim report
