Week 1 HW: Final Project Ideas

(1) What you would like to biomanufacture and why?

Project: Sensing α-Synuclein-Driven Mitochondrial Proteostatic Failure in Parkinson’s Disease (PD) with a ClpP Activity-Linked Synthetic Promoter Circuit (gain of signal upon stress)

Goal: Design a ClpP activity-linked synthetic promoter circuit that exploits a known ClpP-specific degron fused to a transcription factor that would drive the expression of a reporter in human dopaminergic neurons.

Background: ClpP is a mitochondrial matrix protease that degrades misfolded or damaged proteins and has recently been shown to be inhibited by α-synuclein (through direct binding at the NAC domain of ClpP, Hu et al., 2025), representing a novel mechanistic link between α-synuclein pathology and mitochondrial proteostatic failure in Parkinson’s disease.

Problem: there is no PD-specific marker of mitochondrial stress

How the circuit works: Developing PD (low α-synuclein) -> still active ClpP -> active TF -> reporter OFF Disease progression (accumulated a-synuclein) -> inactive ClpP -> TF degraded -> reporter ON (gain of fluorescent signal)

Significance: The tool can be used as a readout and signal mitochondrial stress (early or late?) in PD (in human neurons), tested in high-throughput platforms using patient cells (dopaminergic neurons and brain organoids).

References: Hu, D., Sun, X., & Qi, X. (2025). Disrupting α-Synuclein–ClpP interaction restores mitochondrial function and attenuates neuropathology in Parkinson’s disease models. Molecular Neurodegeneration, 20(1), 126.

(2) What living system would you use?

Mammalian cell lines (e.g., HEK293 for initial validation and later iPSC-derived dopaminergic neurons in brain organoids supplying the full disease-relevant context) that naturally contain ClpP can be used to produce the circuit. A transcription factor fused to a degron and a reporter needs to be introduced into the cells to react with endogenous ClpP.

Transfection or viral transduction would introduce a DNA construct encoding the TF-degron fusion protein and the reporter gene under a synthetic promoter. The cells will be supplied with a culture medium and gases. The genetic construct and the components introduced with the media needed for cell metabolism will allow continuous transcription and translation of the TF-degron fusion. When proteolysis is inhibited, TF-degron would escape degradation by the mitochondrial ClpP protease (inhibited by a-synuclein binding the protease), accumulate, and drive the expression of the reporter, signalling proteolytic stress and the disease progression. TF-degron would bind to a promoter of the reporter gene and the cell would produce the reporter protein. Overall, the cells are manufacturing the sensor responsive of endogenous protease activity, and the same cells are generating the signal.

(3) How would you use automation?

A fully automated, remotely operated research laboratory with robots and automated instruments like Cloud Lab or Opentrons can be used for the initial circuit assembly in order to save time on the whole workflow (assembly, sequencing, transfection) and test different variations of the circuit in parallel. Next, handling mammalian cells and organoids can be automated in robotic cell culture platforms or automated bioreactors to compensate for or eliminate variability of cell culture and developmental variability of brain organoids. Nuclera automated microfluidics platform can be used to test different concentrations of α-synuclein (and the consequential inhibition of ClpP) that would mimic the disease progression. In the case of brain organoids, high-content imaging (spinning disc confocal or light-sheet) needs to be automated to measure the reporter expression repeatedly across many conditions. There could be solutions for automated data analysis to have efficient design-test-learn loops for iteration.

(4) How much would you need to make, and how long would it take?

For my final product (an organoid expressing the circuit with a sensor and a reporter), I’d need a quantity that would simply be publishable. For that, experimental conditions would need to be defined. Those would include a control, different levels of α-synuclein and ClpP, several PD-relevant mutations. The variability has to be established to calculate the number of biological replicates for each condition. I’d also need technical replicates. The number of conditions would need to be multiplied by the biological and technical replicates to define the number of organoids needed. Some additional samples must be planned to compensate for a failed phenotype or unsuccessful construct delivery.

For the design and validation phase, when the target product is different, different calculations are needed. For Cloud Lab, the number of circuit variants needs to be determined, as well as the number of reactions needed for assembly, the final amount of DNA that needs to be produced and transfected, the number of sequencing reactions. For opentrons, the unit will probably be a well plate with the target cells. That would depend on the number of circuit variations produced with Cloud Lab and the number of replicates decided. For Nuclera, I’d need to define the number of conditions that I’m testing and the number of replicates.

For the Cloud Lab and manufacturing the plasmids as the first step, the how long depends on assembly time, transformation, colony growth, sequencing time for one construct variant. Plus circuit design verification time, shipping time, the number of parallels possible, internal schedule. The DNA yield for each variant must be compatible with the amount needed on the Opentron step (depending on the number of cells in the well and the number of replicates).

The first living system (the test one) would be mammalian cells (e.g. HEK cells) in plates handled by Opentrons. If e.g. 20 construct variants produced with Cloud Lab need to be tested (that would include several degrons, several TFs, and promoters), the number of replicates needs to be decided. The number of variants multiplied by replicates will define the number of wells. The workflow may include just one plate, and manufacture at scale is not needed.

(5) What I don’t know yet

  • would only the known degron be needed for the project or some specific variants could be engineered to be effectively recognised by ClpP
  • can a circuit be engineered so that the TF fused to a degron (or some other design) could be delivered to mitochondria, where ClpP would recognise the degron (or where another sensor would respond to a failing ClpP enzyme).
  • can a degron be engineered in a way that it’s exposed to ClpP rather than buried inside the TF
  • would the chosen TF work efficiently while being fused to degron variants
  • the calculations, including units, assumptions, and sources for important numbers
  • the number of construct variants
  • the whole set of experimental conditions
  • the number of biological and technical replicates for each step
  • how long brain organoids would need to grow to show relevant inhibition of ClpP leading to reporter expression
  • ..
  • ..