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

哺乳动物细胞中新颖的逻辑门可以扩大合成生物学的范围，造福社会。合成生物学致力于构建用于研究、工程和医学应用的人工生物系统。逻辑门通常是这些应用的基础。例如，合成生物学家利用逻辑门构建了基于细胞的传感器，用于检测环境污染物、有毒化学物质、病原体、癌细胞等，所有这些都对社会有直接的好处。此外，逻辑门还可以用来开发新的疗法。事实上，医学应用是合成生物学最令人兴奋的领域之一。我们希望在这里进行的研究是在哺乳动物细胞中设计新的逻辑门。这些逻辑门将处理输入信号，以在哺乳动物细胞中产生输出信号。这些信号是二进制的(例如是或否)。在我们的设计中，输入信号将是小分子的存在或不存在，否则这些小分子对细胞没有影响。这一特性很重要，因此分子可以仅用于控制逻辑门输出，而不会干扰任何细胞过程。在我们的设计中，输出信号将是蛋白质的功能或非功能信号。我们希望设计的逻辑门能够快速处理输入信号，并根据输入信号的变化相应地调整其输出信号，就像一台简单的小型计算机。这些特点意味着，建议的逻辑门将特别适用于需要对蛋白质功能或细胞事件进行可逆的精细调节，但现有技术难以实现的应用。我们将使用自然界中不存在的氨基酸作为输入信号的小分子。自然界使用20种氨基酸作为构建我们身体蛋白质的基础。在这里，我们打算使用非天然氨基酸，这些氨基酸无毒，对细胞没有明显影响。更重要的是，使用我们专业的特殊技术，这些非天然氨基酸可以根据我们的意愿插入到蛋白质的特定位置。我们已经使用这项技术来控制蛋白质的功能和基因编辑，通过存在或不存在非天然氨基酸。然而，这种技术从未被应用于逻辑门工程。因此，建议的逻辑门是新颖的，也将是对现有的逻辑门的补充。因此，使用不同的逻辑门组装复杂的遗传电路是可能的。在这个项目中，我们将设计执行不同逻辑操作的基本逻辑门。这些基本逻辑门可以组合起来执行复杂的任务，并且是更复杂的逻辑门的基础。我们将在哺乳动物细胞中构建它们，并使用不同的分析技术来表征它们的性能。我们还将实现一个逻辑门来控制工程免疫细胞的功能。免疫细胞是我们身体防御系统的一部分。它们可以被改造成对抗非传染性疾病，比如癌症。这种基于细胞的疗法很有前途，NHS为患有B细胞急性淋巴细胞性白血病的儿童和年轻人提供了这种疗法。在某些情况下，基于细胞的疗法甚至治愈了所有其他疗法都失败的人。然而，它们也可能导致不良副作用，甚至病人死亡。虽然生物学研究和医学应用不在本提案的范围内，但这里将要开发的逻辑门可以提高当前基于细胞的治疗的安全性，解决医生和患者的一个关键问题。总体而言，我们建议在哺乳动物细胞中设计新的逻辑门。这些逻辑门将对无毒的非天然氨基酸做出反应，并可用于蛋白质功能或细胞属性的可逆精细调节。所提出的逻辑门在不同的生物医学应用中具有潜力，可能会对社会产生直接的好处。

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.