Novel logic gates in mammalian cells based on genetically incorporated unnatural amino acids

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Novel logic gates in mammalian cells can expand the scope of synthetic biology, benefiting the society. Synthetic biology focus on building artificial biological systems for research, engineering and medical applications. Logic gates are often the foundation of these applications. For example, synthetic biologists have used logic gates to construct cell-based sensors for detecting environmental pollutants, toxic chemicals, pathogens, cancer cells, etc. All of which have direct benefit to the society. In addition, logic gates can also be used to develop novel therapeutics. In fact, medical application represents one of the most exciting areas for synthetic biology. The research we wish to carry out here is to engineer novel logic gates in mammalian cells. These logic gates will process the input signals to produce an output signal in mammalian cells. These signals are binary (e.g. yes or no). In our design, the input signals will be the presence or absence of small molecules that otherwise have no effects to the cells. This characteristic is important so that the molecules can be used solely to control the logic gate output without interfering any cellular processes. In our design, the output signal will be functional or not of a protein. We wish to engineer logic gates that process the input signals rapidly and adjust its output signal accordingly upon change of the input signals, like a simple small computer. These features mean that the proposed logic gates will be particularly useful for applications where reversible fine regulation of a protein function or a cellular event is required but difficult to achieve by existing technologies.We will use amino acids that do not exist in nature as the small molecules for the input signals. Nature uses 20 amino acids as the building blocks to construct proteins in our body. Here, we intend to use unnatural amino acids that are not toxic and pose no observable effects to cells. More importantly, using a special technique of our expertise, these unnatural amino acids can be inserted into specific position of a protein at our wish. We have used this technique to control protein function and gene editing by the presence or absence of an unnatural amino acid. However, this technique has never been applied for logic gate engineering. The proposed logic gates are thus novel and will also be complementary to those currently available. It is therefore possible to assemble complex genetic circuits using different logic gates. In this project, we will engineer the basic logic gates that perform different logical operations. These basic logic gates can be combined to perform sophisticated tasks and are the basis of more complex logic gates. We will construct them in mammalian cells and characterise their performance using different analytical techniques. We will also implement a logic gate to control the function of engineered immune cells. Immune cells are part of our body's defence system. They can be engineered to combat non-infectious diseases, like cancer. Such cell-based therapies are of great promise and are provided by the NHS for children and young people with B cell acute lymphoblastic leukaemia. In some case, the cell-based therapies have even cured people where all other treatments have failed. However, they could also cause adverse side effects and even patient death. Although biological investigations and medical applications are outside the scope of present proposal, the logic gates to be developed here could improve the safety of current cell-based therapies, addressing a key concern of doctors and patients. Overall, we propose to engineer novel logic gates in mammalian cells. These logic gates will respond to non-toxic unnatural amino acid and can be used for reversible fine regulation of a protein function or a cellular property. The proposed logic gates have potential in different biomedical application and will likely have direct benefit to the society.