Individual Final Project: PARP1-HPF1 Split-GFP Biosensor

A Cell-Free Split-GFP Biosensor for the PARP1-HPF1 Interaction

Student: Constantin Convalexius

Node: Lifefabs Institute

Course: HTGAA 2026

Project type: DNA construct design + cell-free expression + protein-protein interaction biosensor

Wet-lab scope: 3 Twist clonal genes + 1 Ginkgo Cloud Lab cell-free expression assay

One-Sentence Summary

I am building a cell-free split-GFP biosensor to test whether a PARP1 catalytic-domain construct and its partner HPF1 can be co-expressed in an E. coli cell-free system and generate green fluorescence when the two proteins interact.

The Honest Scope

This project is not a full rejuvenation experiment. It does not directly measure cellular reprogramming, epigenetic age, PARP1 catalytic activity, or gene-regulatory changes in living cells.

The realistic experiment I can run through HTGAA, Twist, and Ginkgo Cloud Lab is narrower and cleaner:

Can I design and build a three-construct split-GFP biosensor that reports the PARP1-HPF1 interaction in a cell-free reaction?

That is still valuable. Before testing a large biological hypothesis in mammalian cells, I first need a working molecular tool. This project builds that tool.

Abstract

Partial cellular reprogramming can reverse some molecular features of aging, but the mechanisms that separate rejuvenation from loss of cell identity remain incompletely understood. Recent work by Yücel et al. identified conserved master regulators associated with reprogramming-induced rejuvenation, including EZH2 and PARP1. One striking observation is that the catalytically dead EZH2-Y726D mutant can still support rejuvenation-associated effects, suggesting that some regulators may act through non-canonical structural or scaffolding roles rather than only through enzyme activity.

My final project builds a practical experimental tool to begin studying that idea in a remote, HTGAA-compatible format. Instead of attempting a full mammalian reprogramming experiment, which would require cell culture, sequencing, and a much larger budget, I focus on one molecular interaction: PARP1 and HPF1. HPF1 is a known binding partner of the PARP1 catalytic domain and helps direct PARP1-dependent ADP-ribosylation biology. I designed three Twist clonal gene constructs: PARP1 catalytic domain wild type fused to GFP11, PARP1 catalytic domain E988K mutant fused to GFP11, and full-length HPF1 fused to GFP1-10. These constructs are designed for E. coli cell-free protein synthesis at Ginkgo Cloud Lab.

The broad objective is to create a working cell-free split-GFP biosensor for the PARP1-HPF1 interaction. My hypothesis is that co-expression of HPF1-GFP1-10 with PARP1cat-GFP11 will produce fluorescence above background if the proteins bind and bring the split-GFP fragments together. The expected outcome is not a direct reprogramming result, but a validated construct-and-assay pipeline that can be expanded later to more regulators and more rigorous functional assays.

Why This Is HTGAA-Specific

The biological motivation comes from rejuvenation and reprogramming literature, but the HTGAA contribution is the engineering pipeline:

• I designed custom DNA constructs.
• I used Twist Bioscience to turn those designs into physical plasmids.
• I designed the experiment around Ginkgo Cloud Lab cell-free expression instead of local wet-lab access.
• I built a minimal fluorescence biosensor readout that can be executed remotely.
• I am documenting the design-build-test-learn cycle honestly, including what the assay cannot prove.

This is the HTGAA part: taking a biological idea and turning it into a buildable synthetic biology experiment.

Background

The Big Biological Motivation

Yücel et al. (2025) reconstructed gene regulatory networks across partial reprogramming systems and identified conserved regulators associated with rejuvenation. A key observation motivating my project is that EZH2-Y726D, a catalytically impaired EZH2 mutant, can still support rejuvenation-associated effects. This suggests that at least some reprogramming regulators may have important non-canonical roles beyond their classic enzymatic activity.

PARP1 is another regulator in this general biological space. PARP1 is best known as a DNA damage response protein and poly(ADP-ribose) polymerase. Its catalytic activity uses NAD+ to build ADP-ribose chains on target proteins. However, PARP1 also participates in protein complexes, which makes it a good candidate for asking whether molecular interactions can be separated from enzymatic activity.

Why HPF1?

HPF1 stands for Histone PARylation Factor 1. It directly interacts with the PARP1 catalytic domain and changes how PARP1 modifies proteins. This makes HPF1 a useful partner for a simple biosensor: if PARP1 and HPF1 bind in the cell-free reaction, split GFP may reassemble and produce green fluorescence.

Why Split GFP?

GFP is the green fluorescent protein. In split-GFP systems, GFP is divided into two pieces:

• GFP1-10: a large fragment that is not strongly fluorescent by itself.
• GFP11: a small peptide fragment that is also not fluorescent by itself.

If two proteins bring GFP1-10 and GFP11 close together, the GFP barrel can reassemble and become fluorescent. In my design, HPF1 carries GFP1-10 and PARP1 carries GFP11. Fluorescence therefore becomes a proxy for PARP1-HPF1 proximity.

Project Aims

Aim 1: Build the Biosensor

The first aim of my final project is to build and test a cell-free split-GFP biosensor for the PARP1-HPF1 interaction by using DNA construct design, Twist clonal gene synthesis, E. coli codon optimization, and Ginkgo Cloud Lab cell-free protein expression.

Aim 2: Add Biochemical Controls Later

If the biosensor works, the next step is to add biochemical controls that distinguish binding from catalytic activity. This would require a PARP1 enzymatic activity assay, such as NAD+ depletion or PARylation detection, and expression quality control such as Echo-MS or SDS-PAGE.

Aim 3: Scale to More Regulators

The long-term vision is to create a panel of cell-free biosensors for conserved reprogramming regulators. Each biosensor would test a specific protein-protein or protein-DNA interaction and compare wild-type versus catalytic-dead or interaction-altered variants.

Construct Design

I ordered three clonal gene constructs from Twist Bioscience.

Why Use the PARP1 Catalytic Domain Instead of Full-Length PARP1?

Full-length PARP1 is large and multi-domain. Large human proteins can be difficult to express in E. coli cell-free lysate. I therefore use the PARP1 catalytic domain to make the construct more feasible for cell-free expression while keeping the region that interacts with HPF1.

Why Put GFP1-10 on the N-Terminus of HPF1?

HPF1 uses its C-terminal region to interact with PARP1. If I put the large GFP1-10 fragment on the C-terminus of HPF1, it might block the interaction I am trying to measure. Therefore, HPF1 is designed with GFP1-10 on the N-terminus.

Why E988K?

E988 is part of the PARP1 catalytic machinery. The E988K mutant is expected to disrupt catalytic PARP activity. However, this project does not directly test catalytic activity. In this project, E988K is used as a first-pass mutant comparison in the biosensor.

Experimental Workflow

Literature question
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Choose PARP1-HPF1 as a molecular interaction
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Design three fusion-protein constructs
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Order Twist clonal genes in a T7-compatible vector
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        v
Co-express constructs in Ginkgo cell-free reactions
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Measure split-GFP fluorescence in a plate reader
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Ask: does co-expression produce signal above background?

Experimental Design

Cell-Free Expression Conditions

The planned wet-lab assay uses Ginkgo Cloud Lab cell-free protein expression. Each reaction contains cell-free expression mix plus plasmid DNA. The key comparison is two-plasmid co-expression:

• HPF1-GFP1-10 + PARP1cat-WT-GFP11
• HPF1-GFP1-10 + PARP1cat-E988K-GFP11

Controls

Controls are essential because GFP fluorescence can be misleading without them.

Readout

The direct readout is green fluorescence from reconstituted split GFP.

Normalized fluorescence = sample fluorescence - no-DNA background

Mutant retention score = normalized E988K co-expression signal
                         / normalized WT co-expression signal

This score is useful as a first-pass comparison, but it must be interpreted carefully. A lower E988K signal could mean weaker binding, lower expression, worse folding, or worse split-GFP geometry.

What This Experiment Can and Cannot Show

What It Can Show

• Whether my three designed constructs are compatible with a cell-free expression workflow.
• Whether PARP1cat-GFP11 and HPF1-GFP1-10 can generate split-GFP fluorescence when co-expressed.
• Whether the E988K construct gives more, less, or similar fluorescence compared with the WT construct.
• Whether this biosensor design is worth scaling into a larger panel.

What It Cannot Show

• It cannot prove that PARP1-E988K rejuvenates cells.
• It cannot measure reprogramming potential.
• It cannot measure epigenetic age.
• It cannot prove that PARP1 catalytic activity is abolished unless I add a separate enzymatic assay.
• It cannot prove that scaffolding generalizes across all master regulators.
• It cannot reproduce the full nuclear chromatin context of a living human cell.

This distinction is the most important part of the project. My claim is intentionally limited to the data this experiment can actually produce.

Expected Results

If the biosensor works, I expect the WT co-expression condition to produce fluorescence above the no-DNA and single-plasmid controls. That would mean the PARP1cat-GFP11 and HPF1-GFP1-10 fusion proteins can be expressed and can bring split-GFP fragments together.

For E988K, there are two useful outcomes:

• If E988K fluorescence is similar to WT, the mutant construct still supports the biosensor signal in this assay.
• If E988K fluorescence is much lower than WT, the mutation may reduce binding, reduce expression, alter folding, or change split-GFP geometry.

Either result is useful, but neither result alone proves anything about cellular rejuvenation.

Preliminary Validation Already Completed

• Selected PARP1-HPF1 as a feasible molecular interaction.
• Designed three fusion-protein constructs.
• Corrected the project from an over-ambitious mammalian-cell plan to a realistic cell-free plan.
• Corrected vector logic from mammalian expression to T7-compatible E. coli cell-free expression.
• Corrected the partner construct so HPF1 is fused to GFP1-10 on the N-terminus, preserving the HPF1 C-terminal interaction region.
• Submitted / prepared the three-construct Twist order for the final wet-lab version.

Timeline

Techniques Used

• DNA construct design
• Codon optimization for E. coli
• Twist clonal gene ordering
• Split-GFP reporter design
• Cell-free protein expression
• Plate-reader fluorescence measurement
• Protein interaction assay design
• Literature-based experimental planning
• Bioethical reflection and scope control

Ethics and Responsibility

This project has relatively low direct biosafety risk because it uses non-replicating cell-free reactions rather than engineered organisms released into the environment. The constructs encode human protein fragments and are intended for in vitro expression only.

The main ethical responsibility is truthful communication. Aging biology can easily be overhyped. I need to be clear that this project is not an anti-aging treatment, not a rejuvenation result, and not a clinical experiment. It is a molecular biosensor project that could support future mechanistic work.

Another ethical principle is non-maleficence: avoiding harm. In this context, harm could come from overstating weak evidence, especially in a field where people may be vulnerable to exaggerated longevity claims. I will therefore present the project as tool-building and clearly separate direct data from future speculation.

Budget

The project is intentionally small because the available budget is limited. A larger project testing all regulators would require many more constructs and assays.

Future Work

If the biosensor works, the next steps are:

1. Add a direct PARP1 catalytic activity assay.
2. Add expression quality control such as Echo-MS, SDS-PAGE, or Western blot.
3. Test alternative linker lengths and tag placements.
4. Build additional biosensors for other regulators and partners.
5. Eventually move from cell-free biochemistry into mammalian cell assays.

References

• Yücel et al. (2025). Conserved master regulators of reprogramming-induced rejuvenation. bioRxiv 2025.11.27.690899.
• Yang et al. (2023). Chemical reprogramming and EZH2 inhibition context. Cell.
• Suskiewicz et al. (2020). HPF1 completes the PARP active site and directs ADP-ribosylation. Nature.
• Cabantous, S., Terwilliger, T. C., & Waldo, G. S. (2005). Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology.
• UniProt P09874: Human PARP1.
• UniProt Q9NWY4: Human HPF1.
• FPbase: split-GFP / sfGFP1-10 reference sequence.
• Twist Bioscience clonal gene documentation.
• Ginkgo Bioworks Cloud Lab / cell-free expression documentation.

Final Project Claim

The strongest honest claim for this final project is:

I designed and ordered a three-construct, cell-free split-GFP biosensor for the PARP1-HPF1 interaction. The experiment tests whether the engineered constructs can be expressed in Ginkgo Cloud Lab cell-free reactions and whether PARP1-HPF1 proximity can be detected by fluorescence. This is a foundational HTGAA biosensor project and a first step toward future systematic tests of scaffolding mechanisms in reprogramming regulators.