Team:Fudan/Demonstrate
Demonstration
Transmembrane Logic
Sensing and integrating various transmembrane signals is a key aspect of cellular decision making.
For example, activation of CD8+ cells requires co-activation of TCR and CD28 molecules, meanwhile, this activation can be inhibited by the PD-1 pathway(Hui et al., 2017). By abstracting this biological process, we can get: the activation of CD8+ cell = activated TCR AND (activated CD28 NIMPLY activated PD-1). Programming cells with predictable complex transmembrane signal inputs – customized intracellular signal outputs logic relationships are significant for expanding the widespread applications of mammalian cells, such as cellular immunotherapy(Fedorov et al., 2013; Kloss et al., 2013; Roybal et al., 2016a; Roybal et al., 2016b), tissue patterning(Morsut et al., 2016; Toda et al., 2018).
ENABLE: making cells even smarter
The ingenious design of nature makes us amazing, but inspires us to explore new possibilities. By designing the ENABLE (Engineered, Across-membrane, Binary Logic in Eukaryotes) system and achieved the first complete transmembrane binary Boolean logic in mammals, we could make the pupil outdo the master, make cells even smarter.
Sharpening the knife, Optimizing the Receptor
In order to be able to implement a custom multiplexed transmembrane signal input/output relationship, the first condition is that engineering modular receptor to enable it to recognize extracellular signals and transduce them into customized intracellular signals. SynNotch is a transmembrane receptor with high programmability Therefore, we can use thisideal tool to receive extracellular signals and output customized intracellular signals.
Good tools are prerequisite to the successful execution of a job. In order to make SynNotch more suitable as a tool for receiving complex extracellular signals, we optimized it.
Three-layer design paradigm for dual transmembrane signals
The expression of membrane proteins on the cell membrane is limited. Model analysis tells us how to make cells sensitive to the limited signal molecules, and perform function efficiently is the key to make the transmembrane logic gate system work.
Based on this, we developed the ENABLE system with a 3-layers paradigm: Receptor, Amplifier, and Combiner. By adding an intermediate layer, the “Amplifier” layer, we are able to effectively amplify the transmembrane signal, thus enabling our intracellular components to effectively travel logic functions.
The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.
Five systems make Intracellular logic gate possible
In order to be able to receive dual transmembrane signals and produce complete binary Boolean logic, we designed a set of interactive gramma for the interaction between the "Amplifier" layer and the "Combiner" layer within the cell membrane. This grammar consists mainly of the following elements, a transcription system based on the activating-form, silencing-from or NIMPLY-form promoters (the three are collectively referred to as a synthetic transcription factor-promoter pairs based transcription system); intein-based protein in vivo fusion systems, proteolytic enzyme-based protein in vivo destruction systems (the two are collectively referred to as a protein fusion/destruction-based transcription factor modification system).
From design to testing, these five systems can stand the test.
Explore the specifics of the five major systems
Logic gate：appear only after entreaties
Under the efforts, we have successfully realized six (OR, NOR, AND, NAND, IMPLY, NIMPLY) of the eight (we left the XOR and XNOR to be tested in the future) truly binary logics.
We have designed all implementations of transmembrane binary Boolean logic gates. （一个链接到相关页面）。Here, we use the OR gate as a proof of concept for a three-layer paradigm-based transmembrane binary Boolean logic.
Through dynamic and static observations, we can see that the Receiver cell with transmembrane OR gate can be activated by two Sender cells expressing different membrane antigens.
Click here to see how we captured this beautiful fluorescent （又是一个链接）
Abstract
Contact-dependent signaling is critical for multicellular biological events, yet customizing contact-dependent signal transduction between cells remains challenging. Here we have developed the ENABLE toolbox, a complete set of transmembrane binary logic gates. Each gate consists of 3 layers: Receptor, Amplifier, and Combiner. We first optimized synthetic Notch receptors to enable cells to respond to different signals across the membrane reliably. These signals, individually amplified intracellularly by transcription, are further combined for computing. Our engineered zinc finger-based transcription factors perform binary computation and output designed products. In summary, we have combined spatially different signals in mammalian cells, and revealed new potentials for biological oscillators, tissue engineering, cancer treatments, bio-computing, etc. ENABLE is a toolbox for constructing contact-dependent signaling networks in mammals. The 3-layer design principle underlying ENABLE empowers any future development of transmembrane logic circuits, thus contributes a foundational advance to Synthetic Biology.