Week 1 HW: Final Project Ideas
1. What do you want to make?
I want to biomanufacture a pair of orthogonal synthetic cell-adhesion proteins designed for directed synaptogenesis:1SZ-Nrxn1β (presynaptic): β-neurexin scaffold fused to a synthetic SynZip coiled-coil domain. 2SZ-Nlgn1 (postsynaptic): neuroligin-1 scaffold fused to the complementary SynZip domain.
These proteins act as a programmable “molecular Velcro” that preferentially drives the formation of specific synapses between neurons expressing the matching pair, overcoming the random connectivity of native neurexin–neuroligin interactions. Why?
Directed (orthogonal) synaptogenesis is a frontier tool for:
Precise rewiring of neural circuits for regenerative medicine (epilepsy, Parkinson’s, spinal injuries). Building better brain–computer interfaces and neuroprosthetics. Creating controlled multicellular neural architectures for basic research and biological computing.
My long-term motivation is to contribute tools that let us treat neurological disease by reprogramming connectivity rather than just modulating activity. In the shorter term, the immediate product is a research-grade toolkit (DNA constructs + purified proteins + engineered cell lines) that neurobiology and synbio labs can use.
2. What living system could make it?
Primary chassis: Mammalian cells (HEK293 or CHO)
Input: Optimized DNA constructs (codon-optimized, under hSyn or constitutive promoters, with fluorescent tags mCherry/GFP) delivered by lentiviral vectors or transient transfection. Biological process: Transcription → translation → trafficking to the plasma membrane → secretion or surface display of the chimeric adhesion proteins. Output: Membrane-bound or purified soluble versions of the two complementary proteins.
Alternative / complementary systems:
Cell-free systems (e.g., PURExpress or mammalian cell-free lysates) for rapid prototyping and small-scale pure protein production without the complexity of living cells. Microbial (E. coli) for high-yield production of the DNA parts and possibly truncated soluble domains.
Assumption: Full-length transmembrane versions will require eukaryotic post-translational processing, so mammalian or cell-free eukaryotic systems are preferred for the final functional product.
3. How could automation help you make it?
Automation would be used at three critical stages:DNA design & assembly – Desktop liquid-handling robot (Opentrons OT-2) or cloud lab for high-throughput Golden Gate / Gibson assembly of many SynZip linker variants and promoter combinations. Screening & characterization – Automated transfection, culture, and imaging (cloud lab or microfluidics platform such as Nuclera) to quantify cell–cell aggregation and colocalization of fluorescent tags in 96- or 384-well format. Protein production & purification – Automated bioreactor sampling, affinity purification (His/Strep tags), and quality control (SDS-PAGE, Western, binding assays) so that successful variants can be rapidly scaled from µg to mg quantities.
Why automation? The design space of linkers, SynZip pairs, and expression conditions is combinatorial. Manual screening would be too slow; automation turns the DBTL cycle into a high-throughput process.
4. What would it take to make it at a meaningful scale?
(a) How much?Meaningful research scale = enough material for 50 independent research groups to run pilot experiments for one year. Assumption: each lab needs ~2 mg of each purified protein + 10 µg of high-quality plasmid DNA per year.
→ Total target: 100 mg of each protein + 500 µg of each plasmid.
(b) How long?
DNA: With automated assembly + Twist Bioscience synthesis of the initial constructs → 2–3 weeks. Small-scale protein (µg–mg) in HEK293 suspension culture (1–5 L bioreactors) with automated purification: ~4–6 weeks per batch (including transfection, expression, harvest). Parallel production of both proteins in two bioreactors + automation → first research-grade batch in ~8–10 weeks.
For larger future therapeutic scale (grams), we would switch to stable CHO cell lines in 50–200 L bioreactors and continuous perfusion culture — a completely different manufacturing setup from the prototyping phase.
5. What don’t you know yet? (biggest unknowns)
Functional orthogonality in a real neural context – Will the SynZip pair actually drive preferential synapse formation in primary neurons or organoids, or will native interactions still dominate? Expression level, trafficking and toxicity – Do the chimeric proteins reach the membrane at sufficient density without triggering ER stress or altered cell physiology? Binding kinetics and mechanical strength – We can predict affinity in silico (HADDOCK), but we do not yet know the on/off rates or the force the synthetic synapse can withstand under physiological conditions.
These are the key experimental questions that must be answered before the system can move from a research tool to a reliable biomanufactured product.
Assisted by Grok (xAI). Prompt: “Help me frame my undergraduate thesis on synthetic synaptic adhesion proteins (SynZip-neurexin/neuroligin) as a biomanufacturing project answering the four HTBAA questions: what to make, living system, automation, and scale.”