Team:Fudan/Protocols
Our notebook
PCR and subcloning were performed using standard methods. Detailed primer sequences are provided. All constructs were verified by Sanger sequencing.
Cells were cultured in DMEM supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin and 1x GlutaMax (Gibco). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Gibco). Viral packaging, infection and fluorescence-activated cell sorting were performed using standard methods.
Images, unless otherwise indicated, were captured using an inverted epifluorescence microscope (IX-81, Olympus) and a sCMOS camera (pixel size = 0.3222 μm; Zyla 5.5, Andor; 20x objective N.A. 0.75) and were controlled by Micro-Manager software.
All statistical analysis was performed using Prism (Graphpad) and ImageJ. All experiments were independently performed in triplicates; unless otherwise indicated. Images were combined and annotated in Powerpoint for presentation. Representative images are shown.
For practical reasons, all full-length protocols are in Chinese.
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.