Transmembrane receptors can continuously monitor the extracellular milieu, and transduce specific extracellular signals into intracellular responses. Binding of ligands that match the receptors triggers intracellular signaling cascades and ultimately induces transcriptional changes. We found some latest receptors, with some of them sensing and responding to soluble molecules while others sensing and responding to surface-bound molecules. We placed them in our different devices, giving them different roles, and compared their function to determine the most suitable one.
Receptors for surface-bound molecules:
The best-known examples of synthetic receptors that sense and respond to molecules bound to the surface of other cells are the so-called chimeric antigen receptors (CARs). Based on large number of researches and characterization of CARs, we chose it as the core tool to achieve the killing function of the core device.
Our synthetic receptor CARs contained an extracellular scFv trastuzumab (BBa_K3132022) and an intracellular regulatory domain of the NK-cell receptor complex (BBa_K3132021) (figure 1a). CAR-expressing NK cells can detect tumor cells with HER2 on the surface and attack them.
We investigated the anti-tumor potential of the CAR-transduced NK-92 cells by standard 51Cr-release assays.
SynNotch receptors are synthetic receptors improved from native Notch. This kind of receptors are activated by binding to surface-bound antigens on neighboring cells, which then trigger release of a synthetic transcription factor to modulate target gene expression.
Our synthetic receptor synNotch contained an extracellular scFv against antigen HER2 (BBa_K3132005) bound on the surface of tumor cells, a transmembrane NL domain through cytoplasmic (BBa_K3132005) and an intracellular domain which was set as synthetic transcription factor.(figure 1b).
To characterize synNotch, we firstly found two breast cancer cell lines expressing high and low level of HER2 respectively by flow cytometry. Then we tested the response of synNotch with three different transcription factors fused to intracellular segments. The results show that synNotch had high sensitivity to HER2 signals, moreover, we can optimize its function by fusing the most suitable transcription factors to the intracellular domain.(Figure 3)
Receptors for soluble molecules:
Modular extracellular sensor architecture, also known as MESA, is also a kind of designable synthetic receptors. This kind of receptors are made up of two different chains, PC chain and TC chain. After being induced by ligands, the two chains will dimer and interact with each other. One chain is fused to the TF by a protease cleavage site and the other chain contains a TEV protease. After ligand-induced dimerization, the protease will recognize the cleavage site and cut down the TF to regulate gene expression.
Our synthetic receptors MESA contained an extracellular scFv against SunTag (BBa_K3132007) released by core device, a transmembrane domain based on a submitted parts (BBa_K1065102) and an intracellular domain with transcription factor Gal4-VP64 connected to the protease cleavage site.(figure 1c).
Generalized extracellular molecule sensor, also known as GEMS, is generalizable because it could be adapted to sense a wide range of input signals and express genes as the users wish. Like MESA, GEMS contains two functional chains, however, it exists in the form of dimerization. Ligand binding to the receptors is thought to rotate each chain subunit around its own axis then the activated receptor can induce downstream cascade.
Our synthetic receptor GEMS contained an extracellular scFv against suntag ((BBa_K3132018) released by core device, a transmembrane scaffold based on the erythropoietin receptor and an intracellular domain fused to FGFR1, which is able to activate MAPK pathway and regulate gene expression relied on transcription factors PIP-Elk ((BBa_K3132012)(figure 1d).
We did large amount of experience to characterize GEMS and got some satisfactory results. We constructed different lengths of suntags and tested the receptor's response to these labeled proteins. The results show that GEMS had the most sensitivity to 6xsuntag, and well activation of the target gene.(figure5)
Here we collected these four types of synthetic receptors, hoping to construct a toolbox for creating devices that sense and respond to user-defined ligands, capitalizing on native ligand-receptor interactions and ultimately rewiring cascades and regulating user-defined responses. More information can be searched on their pages.
2.Synthetic transcription factor (SynTF) - synthetic promoter (SynPro) pairs
For modular design of synNotch receptors, we kept the functional sequence of the transmembrane domain from α-CD19 Notch (BBa_K3132006) and used anti-HER2 scFV (BBa_K3132005) as the extracellular domain to specifically recognize HER2. However, the intracellular domain, which will be detached from the transmembrane domain and function as transcription factors has some options. We fused four different transcription factors which function to activate expression of target gene as intracellular domain of synNotch and tested their functions to find a most suitable one with high activity and selectivity.
We referred to Fudan's design of these synthetic transcription factor (SynTF) - synthetic promoter (SynPro) pairs in 2017 (http://2017.igem.org/Team:Fudan/Part_Collection). Similarly, the SynTFs contained three core domain: DNA bingding domain, nuclear location sequence and transcription regulating domain. Based on the role synNotch receptors play, we chose VP64 as transcription regulating domain instead of KRAB that Fudan had used and constructed four types of activating-form SynTFs. After detached, these SynTFs will be located into nuclei and recognize its corresponding promoter, then activate expression of targeted genes.
More details about characterization of these transcription factors are on the page of synNotch (BBa_K3132020). As for their regulated promoters, we fused a minimal promoter CMV to the binding domains of each promoter, the design and activity of these promoters are on their pages.
Here we listed these pairs of Biobricks as a part collection below.