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）