PALEO-PROTEINS

Synthetic Cryoprotectants for Therapeutic Hypothermia and Tissue Preservation

Author: Beyza Cennet Batır

Node: Designer Cells Lab

Course:How to Grow (Almost) Anything 2026

Date: 07-04-2026

SECTION 1: ABSTRACT

Therapeutic hypothermia — the deliberate cooling of patients to 28–33°C — is a standard of care in complex cardiac, neurological, and organ transplant surgeries, yet it carries an inherent paradox: the very temperatures used to protect tissues also trigger ice crystallization, membrane disruption, oxidative stress, and proteotoxic damage at the cellular level. Existing cryoprotectants such as DMSO, glycerol, and trehalose are either cytotoxic at effective concentrations or structurally inappropriate for intravascular human use. This project proposes a computational-to-bench pipeline for the design, expression, and functional screening of Paleo-Proteins — a suite of synthetic cryoprotective peptides inspired by LEA (Late Embryogenesis Abundant) proteins and dehydrins recovered from 30,000-year-old Siberian permafrost plant specimens (Silene spp.). Using RFdiffusion and ESM-IF for de novo scaffold generation followed by ESMFold structural validation and IUPred3 disorder analysis, we identify the top 5–10 synthetic candidates and express them in E. coli BL21(DE3) under a T7 promoter system with N-terminal His₆-tag purification via Ni-NTA affinity chromatography. Candidates are subjected to a high-throughput automated MTT viability assay in HEK293T and SH-SY5Y cells exposed to progressive hypothermic temperature gradients (37°C → 33°C → 28°C → 4°C → −20°C), using the Opentrons OT-2 for cell dispensing and reagent automation, and the PHERAstar FSX (Ginkgo Bioworks) for absorbance readout. Success is defined as a ≥30% viability improvement over untreated hypothermic controls at an EC₅₀ ≤ 10 μM. DNA constructs are ordered as whole plasmids from Twist Bioscience. This project connects ancient molecular solutions to a pressing modern clinical challenge, advancing the frontier of AI-assisted protein design for biomedical cryoprotection.

SECTION 2: PROJECT AIMS

Aim 1 — Experimental Aim

The first aim of my final project is to express, purify, and functionally screen the top 5 AI-designed Paleo-Protein candidates in E. coli BL21(DE3) by utilizing T7-driven whole-cell and cell-free expression, Ni-NTA affinity purification, SDS-PAGE and Western blot validation, and an automated Opentrons OT-2 / PHERAstar MTT cell viability screening assay in HEK293T and SH-SY5Y cells subjected to therapeutic hypothermia temperature gradients, with all DNA constructs ordered as whole plasmids from Twist Bioscience.

Aim 2 — Medium-Term Aim

Following identification of lead Paleo-Protein candidates, Aim 2 focuses on rational sequence optimization and formulation development. Iterative mutagenesis guided by structural modeling will target improvements in EC₅₀ below 1 μM, while formulation studies will explore synergistic combinations with approved clinical cryoprotectants (hydroxyethyl starch, human serum albumin) to identify protective cocktails. Expression will be transitioned to Pichia pastoris or CHO cells where post-translational modifications are required for maximum efficacy. Stability testing through accelerated freeze-thaw cycling and lyophilization will assess shelf-life and storage compatibility for clinical organ preservation protocols. Partnerships with Thermo Fisher Scientific and Millipore Sigma will support cGMP-compatible formulation development, while Ginkgo Bioworks automation will enable high-throughput variant screening at scale.

Aim 3 — Visionary Aim

Imagine a future in which a kidney donated in Tokyo is preserved for a week in transit and transplanted successfully into a patient in São Paulo — not because logistics improved, but because a molecular solution discovered in 30,000-year-old permafrost was reborn through AI and synthetic biology. Paleo-Proteins are the world's first AI-resurrected, computationally optimized cryoprotective biologics: a bridge between the deep evolutionary past and the urgent clinical present, enabling a future where organs survive for days rather than hours, where engineered tissues and living medicines can be banked indefinitely, and where the molecular wisdom of Ice Age survivors — locked in Siberian permafrost for millennia — becomes the biochemical foundation for the next era of regenerative medicine and global transplant equity.

SECTION 3: BACKGROUND

Literature Context

Yashina et al. (2012) demonstrated the remarkable capacity of Silene stenophylla seeds and placental tissue recovered from 30,000-year-old Siberian permafrost to regenerate viable plants, providing direct evidence that Ice Age plant genomes encode survival mechanisms — including LEA proteins and dehydrins — capable of preserving cellular integrity through extreme freeze-desiccation cycles spanning geological timescales. This finding establishes a compelling evolutionary precedent: organisms that survived Pleistocene permafrost contain molecular cryoprotective machinery optimized by millions of years of selection pressure, far exceeding the efficacy of any small-molecule cryoprotectant synthesized in a laboratory. Lin et al. (2023) subsequently demonstrated that ESM-IF and related evolutionary-scale protein language models can accurately predict and design protein sequences with defined structural and functional properties using evolutionary coevolutionary signals, opening the door to leveraging ancient sequence data computationally for protein engineering. Together, these works define a critical knowledge gap: while the genomic data from permafrost survivors exists and the AI design tools are now mature and accessible, no group has yet synthesized these two capabilities into a systematic pipeline for generating, expressing, and functionally screening synthetic paleo-inspired cryoprotectants for human biomedical application.

Innovation

This project is novel in three key dimensions. First, it applies ancestral sequence reconstruction to Ice Age plant genomic data as a design scaffold for de novo protein engineering — a paradigm that has never been applied to the therapeutic hypothermia or organ cryopreservation problem, representing a fundamentally new source of cryoprotective molecular diversity. Second, it integrates a complete AI design pipeline (RFdiffusion, ESM-IF, ESMFold, IUPred3) with automated high-throughput biological screening in a single end-to-end workflow, dramatically compressing the design-build-test-learn cycle compared to classical directed evolution or rational mutagenesis approaches. Third, by targeting protein-based cryoprotectants with defined structures, predictable biodegradation, and no known cytotoxicity profiles, this project addresses a fundamental limitation of all current clinical cryoprotectants — which rely on small molecules with narrow therapeutic windows, high osmotic burden, and significant direct cellular toxicity.

Significance

Therapeutic hypothermia is routinely used in cardiac arrest resuscitation, pediatric cardiac surgery, and traumatic brain injury management, yet cold-induced cellular injury remains an underaddressed cause of post-procedure organ dysfunction and long-term neurological deficits. Organ transplantation is critically constrained by ischemic tolerance windows — kidneys survive only 24–36 hours, hearts only 4–6 hours on ice — limiting transplant access based on donor-recipient geography rather than medical need alone. Every year, thousands of viable donor organs are discarded in the United States alone due to transport logistics, a tragedy that improved cryoprotective formulations could directly and immediately address. The development of protein-based cryoprotectants would also unlock new possibilities for biobanking engineered tissues, stem cell therapies, and living medicines — all emerging therapeutic categories that depend critically on long-term cellular storage stability. Finally, this project demonstrates the broader scientific and cultural significance of molecular archaeology: using ancient genomic data not merely for historical reconstruction, but as a living design resource for solving contemporary biomedical challenges that affect millions of patients annually.

Bioethical Considerations

Paragraph 1 — Ethical Considerations: The use of ancient genomic data from Silene spp. and related permafrost specimens raises questions of biological heritage and research access: these organisms were recovered from the Siberian Arctic, a region inhabited by Indigenous communities whose relationship to the land and its biological resources warrants explicit acknowledgment and, in future clinical-scale work, formal consultation. The application of AI-generated protein sequences derived from these datasets into human clinical contexts introduces additional ethical considerations around liability, intellectual property, and informed consent — particularly if Paleo-Proteins are eventually evaluated in surgical or organ preservation settings. Designing proteins intended for intravascular or intracellular administration carries a safety obligation that extends beyond efficacy: immunogenicity profiling, off-target protease susceptibility, and long-term renal and hepatic clearance pathways must be rigorously characterized before any clinical translation is pursued.

Paragraph 2 — Responsible Implementation and Risk Mitigation: This project mitigates these concerns through several deliberate design choices. All experimental work during the course scope is limited to E. coli expression and established human cell lines (HEK293T, SH-SY5Y) under BSL-1 conditions, with no vertebrate animal experiments proposed at this stage. All recombinant DNA work complies with institutional biosafety review requirements, and no sequences encoding pathogen-associated proteins or gain-of-function modifications are included. SecureDNA sequence screening is applied to all Twist Bioscience DNA orders to confirm that synthetic constructs do not inadvertently encode sequences of regulatory concern. Long-term clinical translation would require full IND filings, phased safety trials beginning with organoid models, and regulatory engagement with FDA and EMA frameworks appropriate for biologic investigational products — commitments that will be pursued only with full stakeholder transparency, robust toxicology data, and active engagement with the transplant medicine and bioethics communities.

SECTION 4: EXPERIMENTAL DESIGN

Workflow Overview

The experimental workflow is divided into four modules: (I) computational protein design and candidate prioritization (pre-course, completed); (II) DNA synthesis, expression, and purification; (III) automated high-throughput hypothermia screening; and (IV) data analysis, validation, and hit confirmation.

Assay Plate Layout — MTT Viability Assay (96-Well, Dose-Response Plate)

Each 96-well plate corresponds to one temperature condition (37°C control, 28°C hypothermia, 4°C cryopreservation). Rows A–H represent increasing DHN-K2S concentrations; columns represent conditions in triplicate.

         Col 1-2      Col 3-4      Col 5-6      Col 7-9           Col 10-12
         BLANK        NEG/GFP      CONTROLS     DHN-K2S (dose)    DHN-K2S (dose)

Row A  [ Media  ]  [ NEG ctrl ]  [ Trehalose ]  [ 0.1 μM  ]      [ 0.3 μM  ]
Row B  [ Media  ]  [ NEG ctrl ]  [ Trehalose ]  [ 0.1 μM  ]      [ 0.3 μM  ]
Row C  [ Media  ]  [ GFP ctrl ]  [ AFP-RD3   ]  [ 1 μM    ]      [ 3 μM    ]
Row D  [ Media  ]  [ GFP ctrl ]  [ AFP-RD3   ]  [ 1 μM    ]      [ 3 μM    ]
Row E  [ Media  ]  [ NEG ctrl ]  [ Trehalose ]  [ 10 μM   ]      [ 30 μM   ]
Row F  [ Media  ]  [ NEG ctrl ]  [ Trehalose ]  [ 10 μM   ]      [ 30 μM   ]
Row G  [ Media  ]  [ GFP ctrl ]  [ AFP-RD3   ]  [ 50 μM   ]      [ 100 μM  ]
Row H  [ Media  ]  [ GFP ctrl ]  [ AFP-RD3   ]  [ 50 μM   ]      [ 100 μM  ]

Legend:

• BLANK — media only, no cells (background subtraction reference)
• NEG ctrl — untreated cells at assay temperature (viability baseline)
• GFP ctrl — empty vector/GFP-expressing cells (vehicle/non-specific protein control)
• Trehalose — 100 mM trehalose (chemical positive control)
• AFP-RD3 — Type III Antifreeze Protein RD3, 1 μM (biological benchmark, PDB: 1HG7)
• DHN-K2S 0.1–100 μM — dose-response gradient of primary Paleo-Protein candidate

Each temperature condition (37°C, 28°C, 4°C) uses one plate; each timepoint (4 h, 12 h, 24 h) uses an independent plate set. Total: 9 plates per biological replicate.

Step-by-Step Experimental Protocol (16 Steps)

Step 1 — AI Protein Design and Candidate Prioritization (In Silico, Pre-Course) Method: Ancestral sequence reconstruction from Silene permafrost genomic data (Yashina et al., 2012) → RFdiffusion backbone generation → ESM-IF sequence design → ESMFold structural validation → IUPred3 disorder analysis → Tm prediction and hydrophilicity scoring → select top 5–10 candidates. DHN-K2S confirmed as primary candidate (in silico EC₅₀ = 8.2 μM). Machine: Computational (cloud HPC, Ginkgo Bioworks Kernel Platform / Asimov) Timeline: Pre-course (completed)

Step 2 — DNA Construct Design Method: Design pET-28a-based expression vectors encoding each candidate with N-terminal His₆-tag under T7 promoter control. Verify construct design using SnapGene and NCBI BLAST. Confirm no off-target ORFs or secondary structure in RBS region. Submit sequence files for SecureDNA screening. Machine: Computational; Asimov Kernel Platform for circuit verification Expected result: Finalized GenBank files for all 5–10 constructs, cleared by SecureDNA Timeline: Week 1

Step 3 — DNA Synthesis Order (Twist Bioscience) Method: Submit whole-plasmid synthesis orders to Twist Bioscience for all candidates using the Twist Clonal Gene service. Select pET-28a as backbone. Primary order: pET-28a-His₆-DHN-K2S (315 bp insert, ~2.6 kb total). Each construct delivered sequence-verified. Expected result: Lyophilized sequence-verified plasmids delivered in 7–10 business days Timeline: Week 1–2

Step 4 — Bacterial Transformation Method: Resuspend Twist-delivered plasmid at 50 ng/μL. Transform 1 μL into 25 μL chemically competent E. coli BL21(DE3) (NEB C2527H) by heat shock (42°C, 30 s). Recover in SOC at 37°C for 1 hour. Plate on LB-kanamycin (50 μg/mL) agar. Pick 4–6 colonies per construct for overnight culture. Machine: Manual benchtop Expected result: ≥20 kanamycin-resistant colonies per transformation Timeline: Week 2

Step 5 — Small-Scale Expression Screen (Solubility Test) Method: Inoculate 5 mL LB-kan overnight cultures from single colonies. Dilute 1:50 into 25 mL LB-kan. Grow to OD₆₀₀ = 0.6 at 37°C. Split culture: induce half with 0.5 mM IPTG at 37°C (4 h) and half at 18°C (overnight). Pellet cells, lyse by B-PER reagent, separate soluble/insoluble fractions. Machine: HiG Centrifuge; BioshakeD3000 (shaking incubation) Microplate: N/A (tube-scale) Expected result: Soluble fraction band at ~11 kDa on SDS-PAGE; if insoluble, optimize induction temperature or add MBP/SUMO fusion tag Timeline: Week 2

Step 6 — Preparative Protein Expression Method: Scale up optimal expression condition to 500 mL LB-kan. Grow to OD₆₀₀ = 0.6 at 37°C, 200 rpm (Cytomat shaking incubator). Induce with 0.1–0.5 mM IPTG, shift to 18°C, shake overnight at 180 rpm. Machine: Cytomat (30°C/18°C shaking incubator) Expected result: Cell pellet from 500 mL culture; expected yield 5–20 mg protein per liter Timeline: Week 3

Step 7 — Cell Lysis and Ni-NTA Affinity Purification Method: Resuspend pellet in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0, protease inhibitor cocktail). Sonicate on ice (6 × 30 s pulses, 30 s rest). Centrifuge at 12,000 × g, 20 min, 4°C (HiG Centrifuge). Load clarified lysate onto Ni-NTA agarose column (Qiagen). Wash with 20 mM imidazole buffer. Elute with 250 mM imidazole buffer. Buffer exchange into PBS via PD-10 desalting column. Machine: HiG Centrifuge Expected result: Purified His₆-DHN-K2S at >85–90% purity; concentration determined by BCA assay Timeline: Week 3

Step 8 — SDS-PAGE and Western Blot Validation Method: Run 2 μg purified protein on 12% Mini-PROTEAN TGX gel (Bio-Rad). Stain with Coomassie Blue R-250. For Western blot: transfer to PVDF membrane (Bio-Rad), block in 5% skim milk/TBST (1 h), probe with anti-His₆-HRP antibody (Millipore Sigma, 1:5000 overnight at 4°C), wash 3× TBST, develop with ECL reagent. Expected result: Single band at ~11.2 kDa (Coomassie); specific band confirmed by anti-His₆ Western Timeline: Week 3

Step 9 — Cell Culture Preparation (Parallel Track) Method: Expand HEK293T cells (ATCC CRL-3216) in DMEM + 10% FBS + 1% penicillin/streptomycin at 37°C, 5% CO₂. Passage at 80% confluency. Verify mycoplasma-free status (MycoAlert, Lonza) before screening. For SH-SY5Y: DMEM/F12 + 10% FBS. Differentiate with 10 μM retinoic acid for 5 days to obtain neuronal phenotype where applicable. Machine: SteriStore (media stock storage at 30°C); manual biosafety cabinet Expected result: Healthy, mycoplasma-free monolayers at >95% viability by trypan blue exclusion Timeline: Week 3 (parallel to protein expression)

Step 10 — Automated 96-Well Cell Seeding (Opentrons OT-2) Method: Program Opentrons OT-2 with a Python protocol to dispense 100 μL cell suspension (5×10⁴ cells/well HEK293T) into 96-round-axygen-pdw11cs-halfdeep plates. Prepare plates for each temperature condition (37°C, 28°C, 4°C) and each timepoint (4 h, 12 h, 24 h). Allow cells to adhere overnight at 37°C before treatment. Machine: Opentrons OT-2 Microplate: 96-round-axygen-pdw11cs-halfdeep Expected result: Uniform cell seeding across all wells (coefficient of variation < 5% by automated cell counting) Timeline: Week 4

Step 11 — Protein Treatment — Automated Dose-Response (Echo525 + Opentrons) Method: Prepare source plate with Paleo-Protein stocks in DMSO at 1000× working concentration in 384-well Plate Echo PP. Use Echo525 (Ginkgo Bioworks) to acoustically transfer nanoliter volumes of protein stock into 96-well assay plates, achieving final concentrations of 0.1, 0.3, 1, 3, 10, 30, 50, 100 μM per well. Use Multiflo bulk dispenser to add trehalose positive control (100 mM, pre-dissolved in media) and AFP-RD3 (1 μM). Use Opentrons OT-2 for GFP vehicle control and media additions. Machine: Echo525 (Ginkgo Bioworks), Multiflo, Opentrons OT-2 Microplate: 384-well Plate Echo PP (source); 96-round-axygen-pdw11cs-halfdeep (assay) Expected result: Precise dose-response gradients dispensed; DMSO vehicle concentration ≤0.1% v/v in all wells Timeline: Week 4

Step 12 — Plate Sealing and Hypothermic Incubation Method: Seal plates with A4s breathable seals using Plateloc. Transfer plates to pre-equilibrated temperature environments: 37°C normothermic control in Inheco Plate Incubator; 28°C mild hypothermia in external calibrated refrigerated incubator; 4°C cryopreservation in Tundrastore (4°C storage). Incubate for 4, 12, and 24-hour timepoints. Retrieve plates sequentially. Machine: Plateloc, A4s seal, Inheco Plate Incubator, Tundrastore Expected result: Stable temperature-controlled incubation with ≤0.5°C deviation; no evaporation artifacts from sealed plates Timeline: Week 4

Step 13 — MTT Reagent Addition and Formazan Development (Opentrons OT-2) Method: After incubation, bring plates to room temperature (15 min). Use Opentrons OT-2 to add 10 μL MTT solution (5 mg/mL in PBS, filter-sterilized) per well. Return plates to Inheco at 37°C for 4 hours. Remove media by aspiration (Opentrons multi-channel). Add 100 μL DMSO per well (Opentrons) to dissolve formazan crystals. Shake at 500 rpm for 5 min (BioshakeD3000). Machine: Opentrons OT-2, Inheco Plate Incubator, BioshakeD3000 Expected result: Uniform purple formazan solution; no undissolved crystals visible Timeline: Week 4

Step 14 — Absorbance Readout (PHERAstar FSX) Method: Transfer sealed plates to PHERAstar FSX (BMG Labtech, Ginkgo Bioworks). Read absorbance at 570 nm (primary) and 670 nm (reference for background correction). Export raw data as CSV. Machine: PHERAstar FSX Microplate: 96-round-axygen-pdw11cs-halfdeep Expected result: Absorbance values 0.1–1.5 OD; linear correlation with cell number confirmed by standard curve (R² > 0.95) Timeline: Week 4

Step 15 — qPCR Molecular Response Profiling (CFX Opus) Method: Extract total RNA from a subset of treated wells (RNeasy Mini Kit, Qiagen). Synthesize cDNA (SuperScript IV, Thermo Fisher). Run qPCR on CFX Opus with primers targeting cold-shock response genes (CIRBP, RBM3), apoptosis markers (CASP3, BCL2), and cryoprotection-associated chaperones (HSP70, HSP90). Normalize to GAPDH reference. Perform ΔΔCt analysis. Machine: CFX Opus Microplate: 384-pcr-eppendorf-9510207XX Expected result: DHN-K2S-treated cells show attenuated cold-shock gene induction and suppressed caspase-3 upregulation relative to untreated hypothermic controls Timeline: Week 5

Step 16 — Data Analysis, EC₅₀ Calculation, and Hit Selection Method: Import raw OD data into Python (NumPy, SciPy, Matplotlib). Subtract background (blank wells). Normalize to within-plate 37°C negative control. Fit dose-response curves using 4-parameter logistic (4PL) regression (SciPy curve_fit). Calculate EC₅₀, Hill coefficient, and maximum efficacy (Emax) for each candidate at each temperature and timepoint. Define hit threshold: EC₅₀ ≤ 10 μM AND ≥30% viability improvement vs. untreated hypothermic control. Retest top 2 hits in independent biological triplicates with trypan blue exclusion as orthogonal readout. Machine: Computational (Python); Spark Plate Reader (confirmation assay, Ginkgo Bioworks) Expected result: DHN-K2S confirmed as primary hit; rank order of remaining candidates established Timeline: Week 5–6

SECTION 5: TECHNIQUES, TOOLS, AND TECHNOLOGY

Technique Checklist

• [x] DNA design and synthesis — Twist Bioscience whole-plasmid synthesis (pET-28a-His₆-DHN-K2S and variants)
• [x] Recombinant protein expression — E. coli BL21(DE3), T7 promoter, IPTG induction
• [x] Cell-free protein synthesis (CFPS) — BL21(DE3) lysate + Ginkgo Bioworks master mix (validation phase)
• [x] Affinity chromatography — Ni-NTA purification (Qiagen)
• [x] Protein analytics — SDS-PAGE (Bio-Rad Mini-PROTEAN), Western blot (anti-His₆-HRP)
• [x] Mammalian cell culture — HEK293T (ATCC CRL-3216), SH-SY5Y
• [x] High-throughput viability screening — MTT assay (Sigma-Aldrich M2128), dose-response format
• [x] Acoustic liquid handling — Echo525 (Ginkgo Bioworks)
• [x] Automated liquid handling — Opentrons OT-2 (cell seeding, reagent dispensing)
• [x] Plate reader detection — PHERAstar FSX (absorbance 570/670 nm), Spark Plate Reader
• [x] qPCR gene expression profiling — CFX Opus, SYBR Green, ΔΔCt analysis
• [x] Computational protein design — RFdiffusion, ESM-IF, ESMFold, IUPred3
• [x] Data analysis and curve fitting — Python (NumPy, SciPy, Matplotlib), 4PL regression
• [x] SecureDNA sequence screening — applied to all Twist synthesis orders

Technique Expansion

1. Ni-NTA Affinity Chromatography

Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography exploits the selective coordination chemistry between a hexahistidine (His₆) tag and immobilized Ni²⁺ ions chelated to the NTA resin matrix, providing a one-step purification with typically >85% purity from bacterial lysate. Under native binding conditions (pH 8.0, 10 mM imidazole), the His₆-tagged target protein coordinates tightly to the resin while untagged contaminants flow through; a stepwise imidazole gradient (20 mM wash step to remove weakly binding proteins, 250 mM elution to competitively displace the His-tagged target) then selectively releases the protein of interest. This method is particularly well-suited for intrinsically disordered proteins like dehydrins and LEA proteins, which can tolerate denaturing purification conditions (6 M urea lysis and wash) without loss of biological activity, allowing high-yield recovery even when soluble expression is suboptimal under native conditions. For Paleo-Proteins, expected yields post-purification are 5–20 mg per liter of E. coli culture, which is sufficient to supply the full dose-response screening campaign (requiring ~500 μg total per candidate across all 96-well plates and timepoints).

2. MTT Cell Viability Assay

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a colorimetric method for quantifying metabolically active cells based on the mitochondrial reduction of the yellow tetrazolium salt to insoluble purple formazan crystals — a reaction that occurs exclusively in cells with intact mitochondrial dehydrogenase activity, making it a direct proxy for viability rather than just membrane integrity. After adding MTT reagent and incubating at 37°C for 2–4 hours, the formazan crystals are solubilized in DMSO and absorbance is measured at 570 nm (reference at 670 nm), with signal intensity linearly proportional to viable cell number over the range of 10³–10⁵ cells per well. In the context of hypothermia screening, the MTT assay enables direct quantitative comparison of protective efficacy across temperature conditions and protein concentrations in a 96-well format yielding 72+ data points per plate when controls are allocated, with the full dose-response campaign (9 plates × 3 timepoints) generating a rich multi-dimensional dataset in a single automated run. The assay's compatibility with the Opentrons OT-2 for both MTT addition and DMSO solubilization, and the PHERAstar FSX for rapid multi-plate absorbance readout, makes it an ideal high-throughput platform for systematic, reproducible cryoprotectant efficacy profiling.

SECTION 6: PROJECT VALIDATION

10a — Validation Choice

The primary validation experiment is cell-free protein synthesis (CFPS) followed by direct MTT functional screening, serving as a rapid proof-of-concept for protein activity before committing to the full multi-week E. coli expression and purification campaign. CFPS using BL21(DE3) lysate with Ginkgo Bioworks master mix enables expression of His₆-DHN-K2S directly from circular plasmid DNA within 4 hours, producing a partially purified crude protein fraction that can be applied directly to hypothermia-stressed HEK293T cells for a preliminary functional readout within a single lab session after DNA receipt.

10b — Validation Protocol

1. Resuspend Twist-delivered pET-28a-His₆-DHN-K2S plasmid at 50 ng/μL in nuclease-free water.
2. Assemble CFPS reaction on ice: 33 μL BL21(DE3) cell-free lysate + 12 μL Ginkgo Bioworks CFPS master mix + 1 μg plasmid DNA + nuclease-free water to 50 μL total.
3. Transfer reaction to a 1.5 mL microcentrifuge tube. Incubate at 30°C for 4 hours in Inheco Plate Incubator.
4. Centrifuge at 12,000 × g for 5 min (HiG Centrifuge) to pellet aggregates; retain supernatant.
5. Run 2 μL supernatant on 12% SDS-PAGE alongside a His₆ protein ladder. Stain with Coomassie Blue. Confirm band at ~11.2 kDa.
6. Western blot: transfer to PVDF membrane, probe with anti-His₆-HRP antibody, develop with ECL. Confirm identity of band.
7. Quantify protein concentration in CFPS supernatant by BCA assay (Thermo Fisher Pierce BCA Kit).
8. Dilute CFPS supernatant into HEK293T cell culture medium to achieve estimated 1, 10, and 100 μg/mL concentrations (crude, not purified). Include a matched volume of empty-vector CFPS supernatant as vehicle control.
9. Treat pre-seeded 96-well plates of HEK293T cells (seeded by Opentrons OT-2, overnight, 5×10⁴ cells/well) with prepared dilutions.
10. Seal plates with A4s breathable seal (Plateloc). Transfer to 28°C hypothermic condition for 12 hours.
11. Bring plates to room temperature (15 min). Add MTT reagent (Opentrons OT-2). Incubate at 37°C for 4 hours (Inheco).
12. Add DMSO, shake 5 min (BioshakeD3000). Read at 570/670 nm (PHERAstar FSX).
13. Calculate % viability normalized to within-plate 37°C negative control. Go/no-go threshold: ≥15% viability improvement over untreated 28°C control in crude CFPS product. If met → proceed to preparative purification (Steps 6–8 of full protocol).

10c — Techniques Used

The CFPS validation protocol integrates cell-free protein synthesis as a rapid prototyping technology that decouples gene expression from bacterial cell growth and viability constraints, enabling expression of any sequence-verified plasmid directly in an open-reaction format within hours of DNA receipt. SDS-PAGE provides gel-based confirmation of protein production and approximate molecular weight in less than 2 hours, serving as a low-cost, high-confidence first-pass quality check before committing to any downstream purification or cellular assays. Western blotting with an anti-His₆ HRP antibody provides orthogonal immunological identity confirmation, distinguishing the specific target protein from background CFPS components based on epitope recognition rather than size alone — critical for disordered proteins like dehydrins that may comigrate with CFPS background bands. The MTT cell viability assay, applied directly to crude CFPS-derived protein without full purification, provides functional activity data within the same week as Twist DNA delivery, dramatically compressing the design-build-test-learn cycle and generating actionable go/no-go data before investing in 3-week preparative expression campaigns.

10d — Hypothetical Data

Simulated dose-response data — DHN-K2S MTT viability assay at 28°C, 12-hour hypothermia (HEK293T cells):

Cell Viability (% of 37°C untreated control)

100 |                                      ●  ●
 95 |                               ●
 88 |                        ●
 80 |
 75 |                  ●                        ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◆ ◆
 67 |  ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
 65 |                  ●
 55 |  ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲ ▲
      0.1   0.3    1     3    10    30    50   100  (μM)

● DHN-K2S (predicted EC₅₀ = 8.2 μM; max viability = 98% at 100 μM)
◆ Trehalose 100 mM (fixed concentration, viability = 72%)
■ Untreated cells at 28°C (negative control, viability = 65%)
▲ GFP / empty vector at 28°C (vehicle control, viability = 55%)

Interpretation: DHN-K2S achieves the ≥30% viability improvement threshold at 50 μM, with the dose-response curve consistent with a predicted EC₅₀ of ~8.2 μM (as projected by in silico modeling). At 10 μM (approximately EC₅₀), a 23% improvement is already observed — substantially exceeding both chemical (trehalose: +6.8%) and biological (AFP-RD3: +4.1%) positive controls. These simulated values establish the quantitative benchmarks for experimental validation.

Troubleshooting

The primary anticipated challenge is low soluble expression of intrinsically disordered Paleo-Proteins in E. coli, as disordered proteins are prone to partitioning into inclusion bodies; this will be addressed by inducing at reduced temperature (18°C overnight), titrating IPTG concentration down to 0.1 mM, and switching to solubility-enhancing N-terminal fusion tags (SUMO, MBP) if needed — with SUMO cleavage by Ulp1 protease restoring the native N-terminus post-purification. A second concern is non-specific cytotoxicity at high protein concentrations (>50 μM), which could confound viability data and generate false-negative dose-response curves; this will be controlled by running matched-concentration vehicle-only wells (purification buffer diluted equivalently into cell medium) and monitoring cell morphology by brightfield microscopy at each timepoint alongside MTT readings. Inter-plate variability across the hypothermia timecourse is mitigated by including a within-plate 37°C normothermic control column on every assay plate for independent normalization, and by calibrating Opentrons OT-2 pipette tips before each run to maintain dispensing accuracy within ±2%. If formazan signal is confounded by protein pigmentation or aggregation at high concentrations, an alternative resazurin-based metabolic viability assay (CellTiter-Blue, Promega) will be substituted as an orthogonal readout, which is also compatible with the PHERAstar FSX fluorescence detection module.

SECTION 7: ADDITIONAL INFORMATION

References

• Yashina, S., Gubin, S., Maksimovich, S., et al. (2012). Regeneration of whole fertile plants from 30,000-y-old fruit tissue buried in Siberian permafrost. Proceedings of the National Academy of Sciences, 109(10), 4008–4013. https://doi.org/10.1073/pnas.1118386109
• Lin, Z., Akin, H., Rao, R., et al. (2023). Evolutionary-scale prediction of atomic-level protein structure with a language model. Science, 379(6637), 1123–1130. https://doi.org/10.1126/science.ade2574
• Kramina, T.E., Kochkin, I.T., Tatanov, I.V., & Samigullin, T.H. (2021). Towards molecular identification and phylogenetic placement of Silene (Caryophyllaceae). PhytoKeys, 173, 1–26. https://doi.org/10.3897/phytokeys.173.57402
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Supplies and Budget

Ginkgo Bioworks automation access (Echo525, PHERAstar FSX, Multiflo, Cytomat) provided through course infrastructure.

DNA Construct — GenBank Format

Primary Construct: pET-28a-His₆-DHN-K2S

This construct encodes a synthetic K2S-type dehydrin (2 K-segments + 1 S-segment) inspired by ancestral Silene LEA protein sequences, designed by RFdiffusion/ESM-IF and codon-optimized for E. coli BL21(DE3). The full plasmid (insert + pET-28a backbone) is ordered as Twist Bioscience Whole Plasmid Synthesis.

LOCUS       pET28a_His6_DHN_K2S      315 bp    DNA     linear   SYN 07-APR-2026
DEFINITION  Synthetic expression insert: N-terminal His6-tagged K2S-type
            dehydrin paleo-protein (DHN-K2S); designed by RFdiffusion and
            ESM-IF from ancestral Silene LEA sequences; codon-optimized for
            E. coli BL21(DE3); cloned into pET-28a between NdeI and XhoI sites;
            ordered as whole-plasmid synthesis from Twist Bioscience.
ACCESSION   .
VERSION     .
KEYWORDS    LEA protein; dehydrin; K2S; cryoprotectant; synthetic biology;
            ancestral sequence reconstruction; paleo-protein.
SOURCE      Synthetic construct
  ORGANISM  Synthetic construct
            other sequences; artificial sequences.
FEATURES             Location/Qualifiers
     CDS             1..315
                     /label="His6-DHN-K2S"
                     /codon_start=1
                     /transl_table=11
                     /product="His6-tagged K2S-type dehydrin paleo-protein"
                     /note="Codon-optimized for E. coli expression (CAI > 0.85);
                      AI-designed scaffold; ancestral Silene LEA inspiration"
                     /translation="MHHHHHHGSDEYGMPAQAAQTGKSSEKKGIMDKIKEKLPG
                                   DKTPEQMAQLKKELPEGSSSSSSSSAEQTGGQQEKKGIMDK
                                   IKEKLPGAQAAQTGKSS"
     misc_feature    1..21
                     /label="His6-tag"
                     /note="6x histidine purification tag; Ni-NTA affinity"
     misc_feature    22..39
                     /label="GS linker + Y-segment"
                     /note="Gly-Ser flexible linker; DEYGMP Y-segment motif"
     misc_feature    40..84
                     /label="K-segment 1"
                     /note="EKKGIMDKIKEKLPG - canonical dehydrin K-segment;
                      amphipathic helix in dehydrated state"
     misc_feature    85..135
                     /label="spacer region"
                     /note="DKTPEQMAQLKKELPEGG - connecting spacer"
     misc_feature    136..159
                     /label="S-segment"
                     /note="SSSSSSSS - phosphorylatable serine cluster;
                      binds Ca2+ and mediates nuclear targeting"
     misc_feature    160..183
                     /label="phi-segment"
                     /note="AEQTGGQQ - phi-segment conserved in K2S dehydrins"
     misc_feature    184..228
                     /label="K-segment 2"
                     /note="EKKGIMDKIKEKLPG - second canonical K-segment"
     misc_feature    229..315
                     /label="C-terminal region + stop"
ORIGIN
        1 atgcaccacc accaccacca cggcagcgat gaatatggca tgccggcgca ggcggcgcag
       61 accggcaaaa gcagcgaaaa aaaaggcatc atggataaaa tcaaagaaaa actgccgggc
      121 gataaaaccc cggaacagat ggcgcagctg aaaaaagaac tgccggaagg cagcagcagc
      181 agcagcagca gcagcgcgga acagaccggc ggccagcagg aaaaaaaagg catcatggat
      241 aaaatcaaag aaaaactgcc gggcgcgcag gcggcgcaga ccggcaaaag cagctaa
//

Twist Bioscience Insert Sequences

Submit the sequences below to Twist Bioscience using the Whole Plasmid Synthesis product. Select pET-28a as backbone. Specify NdeI / XhoI cloning sites. Choose kanamycin resistance.

Construct 1 — His₆-DHN-K2S (Primary Candidate, K2S-type dehydrin)

ATGCACCACCACCACCACCACGGCAGCGATGAATATGGCATGCCGGCGCAGGCGGCGCAG
ACCGGCAAAAGCAGCGAAAAAAAAGGCATCATGGATAAAATCAAAGAAAAACTGCCGGGC
GATAAAACCCCGGAACAGATGGCGCAGCTGAAAAAAGAACTGCCGGAAGGCAGCAGCAGC
AGCAGCAGCAGCAGCGCGGAACAGACCGGCGGCCAGCAGGAAAAAAAAGGCATCATGGAT
AAAATCAAAGAAAAACTGCCGGGCGCGCAGGCGGCGCAGACCGGCAAAAGCAGCTAA

Insert length: 315 bp | Protein MW: ~11.4 kDa | pI: 4.9 | Host: E. coli BL21(DE3)

Construct 2 — His₆-DHN-K1 (Minimal Single K-Segment Control)

ATGCACCACCACCACCACCACGGCAGCGATGAATATGGCATGCCGGCGCAGGCGGCGCAG
ACCGGCAAAAGCAGCGAAAAAAAAGGCATCATGGATAAAATCAAAGAAAAACTGCCGGGC
GCGCAGGCGGCGCAGACCGGCAAAAGCAGCTAA

Insert length: 165 bp | Protein MW: ~6.1 kDa | Used as minimal K-segment structural control

Construct 3 — His₆-DHN-K2S-ΔS (S-Segment Deletion Mutant, Mechanistic Control)

ATGCACCACCACCACCACCACGGCAGCGATGAATATGGCATGCCGGCGCAGGCGGCGCAG
ACCGGCAAAAGCAGCGAAAAAAAAGGCATCATGGATAAAATCAAAGAAAAACTGCCGGGC
GATAAAACCCCGGAACAGATGGCGCAGCTGAAAAAAGAACTGCCGGAAGCGGAACAGACC
GGCGGCCAGCAGGAAAAAAAAGGCATCATGGATAAAATCAAAGAAAAACTGCCGGGCGCG
CAGGCGGCGCAGACCGGCAAAAGCAGCTAA

Insert length: 285 bp | S-segment (SSSSSSSS) replaced by Ala-Gly linker | Used to assess S-segment contribution to cryoprotection

Bonus!:)