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Project Description

My goal is to develop a system capable of converting carbon dioxide into more chemically inert compounds as a means of combating carbon pollution via carbon capture. The system at a baseline should function in two parts: efficient conversion of carbon dioxide into bicarbonate, and conversion of bicarbonate into chemically locked compounds for storage or other practical usage. By utilizing enzymes like carbonic anhydrase to speed up the process, this system could contend with other methods of carbon capture technology or see application as a sustainable, carbon dioxide scrubbing system. Continued iteration would hopefully result in a completely self-replenishing, stable system capable of fixing CO2 with minimal intervention.

Aim 1: Experimental Aim

My goal for this project is to be able to successfully link the two systems together as a proof of concept. I will use cell-free protein expression to produce the enzymes carbonic anhydrase, phosphoenolpyruvate carboxylase, and malate dehydrogenase. I will combine carbonic anhydrase in a solution containing calcium ions and inorganic dissolved carbon dioxide from a sodium derivative, and then measure calcium bicarbonate precipitate output as a proxy for carbonic anhydrase mineralization capabilities. A secondary experiment will be performed using my “Sodastream” to directly dissolve CO2 into the liquid (gaseous carbon dioxide source) as functional evidence for CO2 emission elimination. Secondly, carbonic anhydrase, phosphoenolpyruvate carboxylase, and malate dehydrogenase will be combined in solution without calcium ions, allowing for bicarbonate incorporation into catalyzed reactions to produce malate. Readout will be determined by NADH utilization, which can be measured with a spectrophotometer (NADH is consumed by malate dehydrogenase). Should time permit, I hope to use LLM tools to try to increase the efficiency and stability of these enzymes.

Aim 2: Development Aim

The key technical limitations of this project arise in achieving a stable system capable of functioning with minimal human intervention. Currently, my proposed mechanism requires serial addition of reagents, and the sequestered output has limited viability and utility. A developmental aim for this project would be to optimize the system mechanics to be functional on a commercially viable scale. For example, the enzymes are not capable of sustained function for more than a few hours, so a secondary aim would be designing more kinetically stable enzymes or a system to cheaply manufacture enzymes at a large scale with long chemical viability so that resupply isn’t a prohibitive barrier. Additionally, optimizing the mechanism to utilize the minimum amount of reagents or to utilize exclusively self-regenerating reagents would push the concept to a practical, commercial scale as well.

Aim 3: Visionary Aim

Once viable, the global application of this system would help combat carbon emissions to aid in the battle against climate change. Ideally, this system would be able to capture the majority of CO2 output from an emission source, preventing the accumulation of more carbon in our atmosphere. From car-exhaust pipes, power plant smoke stacks, and home heating systems, a liquid sequestration system could take on many different carbon emission sources with applications across a variety of industries and products. Finally, creating a cell-free system capable of attacking the source problem could then be applied to attacking the atmospheric cleanup problem by deploying biological machinery across new mediums and scales.

Peer Review Citations

• A study of bacteria producing carbonic anhydrase enzyme for CaCO3 precipitation and soil biocementation
• Development of Integrated System for Biomimetic CO2 Sequestration Using the Enzyme Carbonic Anhydrase
• A cell-free self-replenishing CO2-fixing system (PDF)

Protein Sequence/Genomic Data

Carbonic anhydrase

• UniProt: P00915

• NCBI: NM_001738

• Benchling/Twist: Plasmid Map

  

Phosphoenolpyruvate carboxylase

• UniProt: P00864

• NCBI: X05903

• Benchling/Twist: Plasmid Map

  

Malate dehydrogenase

• UniProt: P40925

• NCBI: D55654

• Benchling/Twist: Plasmid Map

  

Sequence of Events

1. Cell Free Protein Expression

• Check for expression (anti-His tag or SDS-PAGE)
• De-Salt / Buffer Exchanged expressed protein for testing conditions
  • Desalinating Spin Columns
  • Nickel Column Exchange Chromatography (Histidine Tagged Protein)

2. Assays (Mineralization/Fixation)

Mineralization Stock

• 20 mM HEPES, pH 7.5–7.8
• 50–100 mM NaCl
• 10–50 µM ZnCl2

Fixation Stock

• 50 mM HEPES, pH 7.8
• 50 mM KCl
• 5 mM MgCl2
• 0.5–1 mM DTT

3. Reagents

• HEPES
• NaHCO3, CaCl2, MgCl2, KCl, NaCl, ZnCl2
• PEP (phosphoenolpyruvate)
• NADH, DTT
• Pure Water, desalting columns/spin filters
• DH5α Compotent Cells
• LB Medium + Ampicillin
• Cell Free Protein Synthesis from Ginkgo
• Sealed reaction tubes or plate sealing film

4. Instruments

• pH Meter (or litmus paper)
• Spectrophotometer (340nm for NADH, 600nm for mineralization)
• 96-Well Plate Reader
• -20ºC Freezer
• 4ºC Frdige
• 30ºC/37ºC Warm Room
• Centrifuge

Mineralization Assay

• Buffer: 20 mM HEPES or Tris (pH 8.2–8.4)
• Substrates: 10–25 mM NaHCO3, 5–15 mM CaCl2
• Enzyme: Carbonic Anhydrase (variable for testing)

Note: pH near 8.3 supports carbonate availability. A non-phosphate buffer avoids false precipitation, and calcium carbonate forms cleanly under these conditions compared to phosphate systems (avoids Ca3PO4 contamination).

NaHCO3 is used first for a simple proof of concept. Further testing with dissolved CO2/carbonated water/gas-equilibrated solution as the carbon source will follow. CA will accelerate hydration and mineralization, providing evidence for capture.

Reaction Conditions

• Full reaction w/ CA
• No Carbonic Anhydrase
• No CaCl2

Readout

• Analyze the change in turbidity at 600nm (expected increase)
• Pellet sample and analyze behavior (CaCO3-like?)
• Relative to controls, Full Reaction with CA should show faster turbidity and/or more precipitate and/or reproducible solid formation

Fixation Assay

CA supplies bicarbonate efficiently from dissolved CO2/bicarbonate pool.

• Reaction Step 1 (PPC): PEP + HCO3− → oxaloacetate + Pi
• Reaction Step 2 (MDH): oxaloacetate + NADH → malate + NAD+

Setup

• 50 mM HEPES, pH 7.8
• 5 mM MgCl2
• 50 mM KCl
• 10 mM NaHCO3
• 2–5 mM PEP
• 0.2 mM NADH

Biologics

• Carbonic Anhydrase (CA)
• Phosphoenolpyruvate carboxylase (PPC)
• Malate dehydrogenase (MDH)
• Note: MDH should be at an effective level equal to or higher than PPC, as PPC is the bottleneck step.

Protocol Notes

• Test a small matrix of input amounts for desalted CFPS outputs.
• Reactions should be sealed/capped as much as possible.
• Prepare bicarbonate and NADH fresh (protect NADH from light).
• Use fresh PEP.
• Keep calcium completely out of this assay.
• Avoid phosphate buffer.

Reaction Conditions

• Full Reaction: CA + PPC + MDH + Carbon Dioxide Source + PEP + NADH
• No CA
• No PPC
• No MDH

Readout

• MDH consumes NADH → decrease at 340nm

Lab Day 1:

Cell-Free Protein Synthesis

1. Opened the Twist Order and performed calculations to determine the volume to rehydrate the DNA
2. Alloquoted Ultrapure H20 and used a P20 to rehydrate the DNA
3. 10–minute centrifuge step at 10 x 1000 min-1
4. Followed CFPS Protocol from Ginkgo https://cdn.shopify.com/s/files/1/0759/9816/7283/files/cfps-economy-protocol.pdf
5. 10 minute centrifuge step with all cell free reactions before 30º C incubation (4-24hrs)
6. Record reaction start time (9:30pm 5/6/2026) and start 30º C incubation

Buffer Prep

1. Created stocks of pertinent buffers for the mineralization assay
   • 1M MgCl2
   • 1M NaCl
   • 1M KCl
   • 1M CaCl2
   • 69mM ZnCl2

Transformation of Expression Plasmid Into Bacteria for Production

1. Pipetted 1-1.4 ul of DNA into competent DH5a solutions
2. 30 minute incubation on ice
3. 45-second heat shock step on thermocycler set to 42º C
4. 100ul addition of SOC medium
5. Plated on Ampicillin Agar and incubated first at 30ºC and now at 37ºC

Lab Day 2:

SDS Page Gel

1. Alloquoted 1:10 dilutions of CFPS samples with PBS in PCR tubes (20ul total volume)
2. 1:2 dilution with DNAse-free water (1:20 total)
3. 1:2 dilution with LDS Sample Buffer (4X) and DNAse Free Water (1:40)
4. Prepared 1L of 1M SDS-PAGE Buffer solution
5. Pipetted 3ul of each sample into each well, with ladder lanes flanking both sides
   • 1 — Ladder, 2 — CA, 3 — MDH, 4 — PEPC, 5 — negative (lysate), 6 — Ladder
6. Ran the SDS-PAGE gel at 150→50→70 volts for ~1.5hrs until the dye reached the bottom
7. Pried the gel out of the box and rinsed 3 times with dH2O
8. Added enough SimpleBlue SafeStain to coat the entire gel
9. 1 hour staining on tilt-table
10. O/N destaining in ddH2O

Liquid Culture Preparation

1. Retrieved LB+Agar plates from incubation
2. Prepared 3 flasks of LB+Amp at 25ml volumes
3. Inoculated liquid culture with colony from LB+Agar plates for each sample
4. Placed liquid cultures in 37ºC warm room for incubation

Lab Day 3:

SDS–Page Gel Imaging

1. Removed stained gel from the tilt table
2. Imaged both with iPhone camera and gel box

Plasmid Mini-Prep

1. Grabbed liquid cultures from the 37ºC warm room
2. Spun down duplicate 1.5mL samples from each culture
3. Followed Monarch Spin Plasmid Mini-Prep Protocol
4. Eluted with heated elution buffer to make 50ul plasmid mixtures
5. Left in -20C

• Ronan then created CFPS reactions for each of my mini-preps while I was away

Lab Day 4:

Ginkgo Preperation

1. Brought all my reagents to Ginkgo to run my assays
2. Prepared fresh dissolved CO2 and measured pH to determine concentration (~10mM)
3. Prepared fresh PEP, HEPES, and NADH for the reaction series
4. Setup two scripts to run the Fixation Assay and the Mineralization Assay

Mineralization Assay

1. Setup script for 96-Well Plate Reader
2. Initial shake
3. OD600 reading every two minutes for 1 hour
4. Repeated protocol 3 times
5. Data export

Fixation Assay

1. Setup Script for 96-Well Plate Reader
2. Initial Shake
3. 380nm and 340nm absorbance readings every 30 seconds for 3 hours
4. Data export

(Wells E and F were actually ran in A and B on a separate plate)

Results

SDS-Page Gel

The gel results were interesting. The bands that appeared were very faint and some were nearly impossible to capture on camera. Based on visual interpretation (I promise I saw these bands): There were bands present at the expected MW for Carbonic Anhydrase and Malate Dehydrogenase. There were no bands present in the Phosphoenolypyruvate Carboxylase lane and there were control bands present in the Lysate lane. Because there were no bands at all present in the PEPC lane, its very likely that the loaded sample was too dilute so this did not invalidate that PEPC was expressed cell-free. Additionally, given the very faint bands across the entire gel, its likely that they were also quite dilute, which actually supports the data from the Mineralization and Fixation Assays.

Raw Data: https://docs.google.com/spreadsheets/d/1FRLAItoWn6G-NxkBwnQOlPxKhreWfG4uzjCG7PMIM_g/edit?usp=sharing

Mineralization Assay

I was very excited with the results of this assay! Its important to note that conditions A(n) and B(n) differ only in the enzyme added. A’s all had the initial CFPS reaction I ran (the one with finnicky SDS-Page results) and B’s all had the secondary CFPS reaction that was prepared from the plasmids that I minipreped. Conditions n= 1-3 and n= 4-6 differ only in the carbon source, A’s using NaHCO3 and B’s using dissolved CO2 from my Sodastream. B4 was the full reaction with dissolved CO2 and showed exactly what I had hoped: faster formation of calcium precipitate, indicating effective conversion of dissolved CO2 into bicarbonate! A4 yeilded a similar result, though at a diminished level which I hypothesize is due to low protein yeild in the initial CFPS reaction. A1 and B1, again, behaved as expected, and were the same reaction except dissolved CO2 was from NaHCO3 which in solution forms CO2. The 6’s and 3’s both behaved as expected: these were reactions without calcium chloride, so they maintained a constant turbidity as no precipitate was forming (there are some weird step-function looking shapes that are almost definetely experimental artificat. Each incidence happens when the script finished and then resumed again, so the OD calibration might have been effected, but within a 1 hour interval, the value stayed constant). Overall very happy with these results!

Fixation Assay

,

This assay was a little more interesting, but again I was definitely very happy with the overall results! Its important to note that conditions A(n) and B(n) differ only in the enzyme added. A’s all had the initial CFPS reaction I ran (the one with finnicky SDS-Page results) and B’s all had the secondary CFPS reaction that was prepared from the plasmids that I minipreped. Conditions n= 1-4 and n= 5-8 differ only in the carbon source, A’s using NaHCO3 and B’s using dissolved CO2 from my Sodastream. Consumption of NADH should result in a decrease in absorbance at 340nm. This should be the case in wells n=1,2,5, and 6. 1 and 5 are full reaction conditions, and 2 and 6 are missing only Carbonic Anhydrase which is a catalyst that increases HCO3 availability in the reaciton, but is not necessary for the reaction cascade to proceed. Other conditions removed PEPC or MDH, and both are necessary for NADH consuption to occur. The way the results played out are as follows: The conditions yeilded expected results for the functional, experimental groups but in weird ways. The B’s had the biggest initial absorbance then decline, where as the A’s had a smaller initial absorbance followed by their decline. However, the other groups also featured a decline in abosorbance over time, but strangely. NADH is unstable so there was likely oxidation and reduction of other substances within the reaction that caused the decline in NADH as it became NAD+. However, looking at the data with all wells, the 1,2,5,6 samples are the cluster that are the lowest on the graph! And after running some statistics across the conditions (comparing experimental to constants (A1 vs A3, A1 vs A4, etc.)) the rate and total absorbance were statistically significant (p < 10^-40) and the normalized rates and absorbances were also significant to the same degree. So despite the weird curves, there was a significant, measurable difference in NADH consumption, indicating that the reactions were successful, and CO2 was being fixed into malate!

Summary

Overall I had a really incredible experience working on my final project and through this class in general. I want to give special thanks to Ronan who supported me through long lab hours and even while I was away to make this final project come together. I am excited and proud to say that my final project was a success!

1. I designed 3 distinct plasmids with custom t7 promoters and 7x Histidines tags that were successfully created
2. The proteins I selected are very complex and large, and yet were expressed cell-free which is remarkable!
3. My assays provided effective proof of concept: measurable CO2 conversion into HCO3 and measurable fixation into 4-C Malate

With these results I would be eager to further explore making this system a reality. I assayed across one set of buffer conditions and can experiment with adjusting the buffer conditions to find an optimal rate for the system! I assayed across one temperature and can add that variable into testing. I used the enzymes in their wildtype form and can now try and optimize them to increase stablity, efficiency, and availability with language models! This project has opened the door for what is possible with biology and cell-free reactions and I am excited to continue doing work in this field.

Group Final Project