Week 1 HW: 🧬 Automated Cell-Free Biomanufacturing of an Insulin Aptamer

Automated Cell-Free Biomanufacturing of an Insulin Aptamer for Screening Insulin After Refrigeration Failure

Core idea: Produce an insulin-binding DNA aptamer using a cell-free system and use it in a disposable electrochemical cartridge to rapidly test insulin samples after a refrigerator or cold chain has failed.

Figure 1. Three-dimensional cartoon representation of insulin (PDB 1TRZ). Image by Diego Mariano, Wikimedia Commons, licensed under CC BY-SA 4.0.

🎯 1. What would you like to biomanufacture and why?

Target product: an insulin-binding DNA aptamer, to be used as the recognition element in an electrochemical sensor.

The main purpose of the device would be screening insulin after refrigeration failure. Insulin requires controlled storage, so a refrigerator malfunction, power outage, or other cold-chain failure can create uncertainty about whether stored insulin should still be used. This idea came to me as a result of my personal experience living in Egypt, where during the summer, frequent blackouts occur to conserve energy. If these blackouts are prolonged, refrigerated medicines are at risk of going bad, and there’s no way to identify what went wrong. I chose insulin as it’s a medication that many of my friends and loved ones rely on for their well-being.

I therefore propose a small, disposable cartridge that rapidly measures insulin in a stored sample after suspected temperature change. The result would be used as a screening step to identify samples that may require further investigation or confirmatory testing.

The device would not initially replace validated pharmaceutical quality-control or potency testing. Instead, it would be designed as a rapid screening tool that could help identify potentially compromised medication for further analysis, and to avoid using them until confirmation of their potency is obtained.

Why an aptamer?

Aptamers are short nucleic-acid molecules that bind specific targets. In this project, the aptamer would recognize insulin, while the electrode converts the binding event into a measurable electrical signal.

🧪 2. What living system would you use?

I would use a cell-free system.

A cell-free system makes sense because the product is a DNA aptamer, so living cells are not required to produce it. The reaction could contain:

• a circular DNA template containing the aptamer sequence;
• a primer;
• DNA polymerase;
• dNTPs; and
• reaction buffer.

The DNA polymerase amplification reaction would generate many copies of the aptamer sequence, which could then be processed and purified.

Circular aptamer template
        ↓
DNA-polymerase amplification system
        ↓
  Multiple aptamer copies
        ↓
 Processing + purification
        ↓
 Aptamer-functionalized electrode
        ↓
 Insulin sample after suspected
 refrigeration failure
        ↓
   Electrochemical signal

The purified aptamer would be attached to a gold electrode in a disposable cartridge. Insulin binding changes the aptamer structure, which can be converted into an electrochemical signal. Published insulin aptasensors have used this general approach. The aptamer would be manufactured in bulk before cartridge assembly, rather than produced during every individual test.

Proposed cartridge architecture

┌────────────────────────────────────────────────┐
│          INSULIN COLD-CHAIN SCREENING          │
│                   CARTRIDGE                    │
├────────────────────────────────────────────────┤
│ Sample inlet                                   │
│      ↓                                         │
│ Sample processing                              │
│      ↓                                         │
│ Aptamer-functionalized gold electrode          │
│      ↓                                         │
│ Insulin recognition                            │
│      ↓                                         │
│ Electrochemical signal                         │
│      ↓                                         │
│ Portable reader                                │
└────────────────────────────────────────────────┘

🤖 3. How would you use automation?

I would use an Opentrons OT-2 to automate the DNA-polymerase amplification system part of the cycle and produce large amounts of aptamer.

The robot could:

• dispense DNA templates and reagents;
• prepare different reaction conditions;
• run replicates and controls; and
• transfer samples for purification and testing.

I believe it would be useful during the development steps, however, I’m unsure about how efficient it would be if this experiment was conducted on a larger scale, perhaps a larger robot?

📏 4. How much would you need to make, and how long would it take?

For a small pilot, I would aim to make enough aptamer for 1,000 disposable screening cartridges.

1. Parameters & Assumptions

• Concentration Ratio: 250 nM aptamer to 10 mircoM insulin (10,000 nM insulin).
  • Source: ScienceDirect Article
  • Ratio: 1 mole of aptamer per 40 moles of insulin (1:40 molar ratio).
• Standard Pen Cartridge Volume: 3.0 mL(0.003 L) per cartridge.
• Standard Insulin Concentration (U-100): 100 units/mL.
  • 1 unit of insulin approx 0.0347 mg.
  • 100 units/mL = 3.47 mg/mL = 3.47 g/L.
  • Molar Mass of Human Insulin: approx 5808 g/mol.
  • Standard Insulin Concentration in Molarity: Concentration = 3.47g/L over 5808 g/mol = 5.974 x 10^(-4) M = 597.4 microM

2. Aptamer Calculations per Cartridge

Using the 1:40 molar ratio of Aptamer to Insulin:

Aptamer Concentration = Insulin Concentration divided by 40= 597.4 microM/ 40 = 14.935 microM = 1.4935 x 10^(-5) mol/L

For 1 cartridge 3.0 mL = 0.003L:

Moles of Aptamer per Cartridge = 1.4935x 10^(-5) mol/L x 0.003L} = 4.48 x 10^(-8) moles (44.8 nmol)

3. Total Requirement for 1,000 Cartridges

Total Moles of Aptamer = 44.8 nmol/cartridge x 1000 cartridges = 44.8 micromol (4.48 x 10^(-5) moles)

Mass Calculation (Assuming average DNA/RNA aptamer molecular weight)

• Assuming an average 40-mer DNA aptamer mass of 12,500 g/mol:

Total Mass of Aptamer = 4.48 x 10^(-5) mol x 12,500 g/mol = 0.56 grams (560 mg)

The exact yield would need to be established experimentally because the amount of DNA produced does not directly indicate how much functional, purified aptamer will be available for the sensor.

Estimated time

⚠️ 5. What don’t you know yet?

1. Can the cartridge distinguish usable from slightly compromised insulin?

This is the most important question for the cold-chain screening application. A sensor could show that insulin is present, but this does not automatically prove that the insulin still has full biological potency. Therefore, I would compare samples stored under acceptable conditions with samples exposed to controlled temperature excursions and compare the sensor results with established analytical or potency measurements.

2. Can the test work with real samples?

Blood or pharmaceutical formulations can contain other substances that may affect the sensor. I would therefore test selectivity, calibration, storage stability, and batch-to-batch variation.

📚 References

1. Yoshida, W., et al. (2009). Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing. Biosensors and Bioelectronics, 24, 1116–1120. https://doi.org/10.1016/j.bios.2008.06.016

2. Wu, Y., Midinov, B., & White, R. J. (2019). Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin. ACS Sensors, 4, 498–503. https://doi.org/10.1021/acssensors.8b01573

3. Zhou, L., et al. (2024). Ratiometric electrochemical aptasensor with strand displacement for insulin detection in blood samples. Analytica Chimica Acta, 1317, 342823. https://doi.org/10.1016/j.aca.2024.342823

4. Karim, A. S., & Jewett, M. C. (2021). Toward sustainable, cell-free biomanufacturing. Current Opinion in Biotechnology, 69, 136–144. https://doi.org/10.1016/j.copbio.2020.12.012

5. Petzold, C. J., & Mukhopadhyay, A. (2025). From bench to biofactory: High-throughput technologies and automated workflows to accelerate biomanufacturing. Current Opinion in Biotechnology, 94, 103320. https://doi.org/10.1016/j.copbio.2025.103320

🤖 AI disclosure

AI client: OpenAI ChatGPT (GPT-5.6 Luna)
Date accessed: September 21, 2026

Prompt used:

“Provide me with the relevant scientific papers for an insulin-detecting aptamer biosensor cartridge, selecting an appropriate microbial, mammalian, or cell-free manufacturing system, describing automation, calculating manufacturing scale and time, and identifying technical unknowns. Write this out as a Markdown file with relevant emojis, Font Awesome Free icons, and a free/openly licensed image relevant to the topic.”

Week 2 HW: 🧬 DNA Design for a Cell-Free Insulin Aptamer

Designing the DNA for Rolling-Circle Manufacturing of an Insulin-Binding Aptamer (IGA3)

Core idea: My Homework #1 product is an insulin-binding DNA aptamer, made in a cell-free system and used in a disposable electrochemical cartridge that screens insulin after refrigeration failure. In this homework, I design the DNA template that would produce it.

Figure 1. Cartoon representation of human insulin (PDB 1TRZ), the target of the aptamer. Image: “Insulina.png” by Diego Mariano, Wikimedia Commons, licensed under CC BY-SA 4.0.

🎯 Part 1: Define the production target

Molecule: IGA3, a single-stranded DNA aptamer that binds human insulin. It is the recognition element of the sensor: it binds insulin on the gold electrode, and the binding event is read out as an electrochemical signal.

Sequence and source: IGA3 was reported by Yoshida et al. (2009), who selected a family of insulin-binding DNA aptamers by in vitro selection [1]. Its sequence is:

5′-GGTGGTGGGGGGGGTTGGTAGGGTGTCTTC-3′   (30 nt)

This sequence can’t be retrieved from a database like UniPort or NCBI gene as it’s a selected molecule rather than a gene, its source is the literature. The sequence is G-rich, which allows it to form a G-quadruplex-like strucutre that’s the reason why it can recognize insulin, for this reason, I’ll be keeping this sequence unchanged and designing everything around it. IGA3-type aptamers have been used in electrochemical insulin sensors. [2, 3].

Chassis: cell-free rolling-circle amplification (RCA) with phi29 DNA polymerase. Since the product is DNA, it requires no transcription or translation, which is why I’ve decided against using a living host.

I chose RCA because it’s isothermal and can continuously amplify a circular DNA template without the repeated heating and cooling cycles required for PCR. phi29 DNA polymerase is well suited to this because it has strong strand-displacement activity and high processivity, allowing it to continuously copy the circular template and generate long DNA products containing tandem repeats of the template sequence [7]. Which is useful for this project because the starting template can contain the IGA3 sequence, allowing RCA to generate many copies of the aptamer from a small amount of circular template. RCA has also been used for high-level amplification of circular DNA templates with phi29 DNA polymerase [7].

Where the product goes: The route is:

Circular template → phi29 RCA → processing into aptamer units
      → purification + QC → attachment to gold electrode → cartridge

The purified aptamer would be immobilized on the electrode of a disposable cartridge, as in published insulin aptasensors [2, 3].

🧩 Part 2: Design the DNA parts

Most of the standard expression parts do not apply as no protein synthesis is required, so I kept only the required parts of a rolling-circle reaction.

Parts used

I am avoiding using a circularization sequence since I don’t know how additional DNA parts would affect the folding of the aptamer into its required structure. The template would be arranged as a single circle containing the parts in this order:

[ primer-binding ] – [ cleavage element ] – [ IGA3 (reverse complement) ] – [ cleavage element ] → closed circle

🔧 Part 5: From design to build

How I would build it. As the circular template seems to be short, I would order it as a single synthetic oligonucleotide and circularize it enzymatically.

Where the other methods fit:

• Microchip and parallel synthesis become useful if I need to test many variants (for example, many different flanking sequences). Tian et al. made gene-length DNA from oligonucleotides made on programmable microchips [4].Kosuri et al. amplified oligonucleotide pools from high-fidelity microchips selectively, then assembled and error-corrected them, which lowers cost per variant [5].

What synthesis constrains. Chemical synthesis is error-prone, and errors accumulate with length. Carr et al. showed that protein-mediated error correction can substantially improve the accuracy of synthetic DNA [6]. However, for this short template purification and sequence verification seem more reasonable than error correction. Important notes concerning this sequence include:

• IGA3 contains a long run of guanines (eight in a row). G-rich sequences can be harder to synthesize and can fold on themselves, so I would check the purity and identity of the synthesized oligonucleotide (for example by mass spectrometry or sequencing).
• As every turn of the circle copies the same template, a mistake in the circle would be amplified into every unit of the RCA product, so I would need to verify the template and sequence the product to make sure there are no mistakes.

⚠️ Part 6: What don’t I know yet?

1. Will RCA-derived IGA3 stay functional?

I do not know whether aptamer made by RCA and then processed binds insulin as well as chemically synthesized IGA3; additionally, nucleotides left at the ends could affect folding. I would compare the two directly: check sequence and size by gel electrophoresis or sequencing, measure insulin binding in a binding assay, and compare the response on the electrode, referencing this to chemically synthesized IGA3.

2. Which helper sequences preserve IGA3 folding?

The primer-binding, junction and cleavage sequences sit next to a G-rich aptamer, so they might pair with it and interfere with its structure. To test this, I would keep IGA3 unchanged and test several designs that differ only in the flanking sequences, first by computational folding prediction and then by insulin-binding assays, in this situation, the parallel synthesis of variants would be useful.

3. How should the RCA concatemer be processed?

To cut the long product into single aptamer units, I would consider enzymatic cleavage at an engineered site or a self-cleaving element, I would need to test both methods, measuring recovery, purity, sequence integrity (are the cut ends correct?), insulin binding and finally sensor performance.

📚 References

1. Yoshida, W., et al. (2009). Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing. Biosensors and Bioelectronics, 24, 1116–1120. https://doi.org/10.1016/j.bios.2008.06.016
2. Wu, Y., Midinov, B., & White, R. J. (2019). Electrochemical Aptamer-Based Sensor for Real-Time Monitoring of Insulin. ACS Sensors, 4, 498–503. https://doi.org/10.1021/acssensors.8b01573
3. Zhou, L., et al. (2024). Ratiometric electrochemical aptasensor with strand displacement for insulin detection in blood samples. Analytica Chimica Acta, 1317, 342823. https://doi.org/10.1016/j.aca.2024.342823
4. Tian, J., et al. (2004). Accurate multiplex gene synthesis from programmable DNA microchips. Nature, 432, 1050–1054. https://doi.org/10.1038/nature03151
5. Kosuri, S., et al. (2010). Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nature Biotechnology, 28, 1295–1299. https://doi.org/10.1038/nbt.1716
6. Carr, P. A., et al. (2004). Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Research, 32, e162. https://doi.org/10.1093/nar/gnh160
7. Dean, F. B., Nelson, J. R., Giesler, T. L., & Lasken, R. S. (2001). Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research, 11, 1095–1099. https://doi.org/10.1101/gr.180501
8. Blondal, T., et al. (2005). Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage. The CircLigase family is used for splint-free circularization of single-stranded nucleic acids. For the practical substrate requirements, consult the CircLigase II protocol

Week 3 HW: 🧪 Cell-Free Protocols

🧪 Section A: Compare Cell-Free Reaction Compositions

🧂 A1. Why do the concentrations of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?

Potassium glutamate functions to reproduce the intracellular environment of E.coli by contributing to the ionic environment. Magnesium glutamate’s role is for magnesium to interact with the negatively charged phosphate groups in nucleic acids, as well as playing a role in the activity + stability of transcription and translation machinery. Despite this, too much magnesium can reduce protein synthesis; therefore, its concentration must be optimized rather than just increased.

The concentrations of potassium and magnesium glutamate differ between compositions, as the optimum depends on the general chemical environment of the reaction, including the lysate, nucleotide concentrations, energy system, and other salts. Changing one component can change the optimum for another component. In fact, Mg-glutamate and K-glutamate have been shown to interact, meaning that the effect of potassium glutamate can depend on the magnesium concentration.

🔎 Which one is more sensitive?

I would consider magnesium glutamate to be more sensitive, as small changes in magnesium availability have a greater effect on transcription and translation compared to small changes in potassium, whose function is mainly to establish the ionic environment.

⚡ A2. How is a PEP + NTP formulation different from a Ribose + NMP formulation?

Their main difference is in how the reaction supplies the energy and nucleotides necessary for transcription and translation.

🔋 PEP + NTP

PEP is phosphoenolpyruvate, which acts as a phosphate source for ATP generation and NTPs are nucleotides such as ATP, CTP, etc.

The ATP regenerated with the aid of PEP is needed to provide energy for protein synthesis, while the NTPs are used for RNA synthesis. The advantages of using PEP + NTP is that it provides a direct source of energy and NTPs, however, it’s costly, as PEP is relatively expensive. PEP can also result in phosphate accumulation during the reaction, Jewett and Swartz observed that PANOx-SP accumulated substantial phosphate during the reaction, which can eventually inhibit CFPS.

🌱 Ribose + NMP

This formulation uses a different strategy, nucleotides are supplied in the cheaper monophosphate form rather than as triphosphates and ribose provides carbon that can be processed by the lysate. The NMPs are converted into their respective triphosphate forms by enzymes already present in the lysate.

Olsen et al. showed that an optimized formulation could produce NTPs from nitrogenous bases and ribose, allowing the system to rely less on expensive preformed nucleotide triphosphates and phosphorylated energy substrates.

🧬 A3. Comparison of two literature cell-free reaction compositions

For this comparison, I chose:

1. PANOx-SP from Jewett & Swartz (2004)
2. RFopt from Olsen et al. (2026)

🔬 Main differences

1. Energy source: PANOx-SP uses 33 mM PEP, whereas RFopt removes PEP and instead uses ribose and glucose. This is one of the most important differences because PEP is an expensive phosphorylated energy substrate. Olsen et al. specifically aimed to eliminate expensive secondary energy substrates while maintaining high protein production.

2. Nucleotide source: PANOx-SP directly provides ATP, CTP, GTP, and UTP. RFopt instead provides AMP, CMP, GMP, and UMP and relies on the lysate’s metabolic machinery to generate the NTPs needed for transcription. This is a major cost-saving strategy because it reduces the dependence on expensive NTP reagents.

3. Magnesium and potassium: PANOx-SP contains 20 mM Mg-glutamate and 175 mM K-glutamate, while RFopt contains 8 mM Mg-glutamate and 362 mM K-glutamate. At first, the RFopt potassium concentration looks high, but this shows that the optimum is specific to the complete reaction composition. Changing the energy and nucleotide systems changes the ionic requirements of the system. Therefore, the optimum salt conditions for one reaction might not be the same for another.

4. Number of components: PANOx-SP contains many conventional components, including PEP, NTPs, putrescine, spermidine, tRNA, folinic acid, NAD, CoA, and oxalate. RFopt was deliberately simplified to reduce the number of costly reagents.

5. Cost versus yield: PANOx-SP was a strong-performing formulation, but its reagent cost was high. Olsen et al. experimentally compared formulations and reported a reagent cost of approximately $4,535/L for PANOx-SP versus $143/L for RFopt, with RFopt producing 2.39 ± 0.28 g/L sfGFP under their test conditions.

📚 References

1. Jewett, M. C., & Swartz, J. R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and Bioengineering, 86(1), 19–26. https://doi.org/10.1002/bit.20026
2. Olsen, M. L., Copeland, C. E., Sundberg, C. A., et al. (2026). Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications, 17, 3478. https://doi.org/10.1038/s41467-026-69605-8
3. Smith, A. A., Wong, E. L., Donovan, R. C., et al. (2026). Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis. bioRxiv. https://doi.org/10.64898/2026.02.05.703998
4. Koch, M., et al. (2021). Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions. https://doi.org/10.1016/j.ymben.2021.08.005

Final Project