Green Chemistry

Contents

Overview

nuCloud utilizes Terminal deoxynucleotidyl transferase to synthesize DNA. Notably, we move towards using enzymes instead of chemical compounds to do the heavy lifting of adding nucleotides into a strand. To further explore the sustainability of this approach, we compared enzymatic and chemical DNA synthesis through an informal life cycle analysis (LCA). LCA is a systematic approach used to evaluate the impacts and efficiency across all stages of a product’s life cycle, from raw material extraction to disposal. LCAs provide critical insights into resource use, waste generation, and potential ecological impacts, thus highlight key areas for improvement and comparison.

Goal and Definition Scope

Goal Definition

The goal of this LCA assessment is to assess the environmental impacts of the life cycle (cradle to grave or cradle to cradle, which is to be determined), process to synthesizing DNA strands, as in the context of biomanufacturnig purposes such as data storage or targeted oligonucleotide therapy. Both phosphoramidite synthesis and enzymatic synthesis will be compared to identify the greener alternative for generating DNA strands for long-term data storage.

System, Functional Unit, and Function of the System

The system object of this assessment of synthesizing DNA strands by two alternative synthesis methods.

The functional unit is the length of DNA obtained, using theoretical stoichmetric amounts of reagents/cofactors that theoretically should produce the same length of DNA.

The function of the system is studied is the potential information storage use of the synthesized DNA strands. The end-of-life scenario of DNA strands is considered to be reusability.

System Boundaries

The boundaries of the system comprise:

  1. for enzymatic synthesis:
    1. production of the enzyme (TdT), according to experimental procedures in Protocols
  2. for chemical synthesis:
    1. deprotection and protection of nucleotides as required for synthesis
  3. for both synthesis methods
    1. reagents, laboratory equipment employed (with EOL)
    2. synthesis of DNA strands

Life Cycle Inventory

Raw Materials

These are the reagents needed for each process.

Chemical

Here are the chemicals needed to perform the major steps of chemical DNA synthesis. Steps for chemical DNA synthesis are from CHEM 413: Bioorganic Chemistry1. Below the hazards and disposal methods are taken from each chemical’s CAS form, and engineering controls are based on member’s experience and harzards listed.

Table 1: Chemicals for Phosphoramidite-based DNA Synthesis
ChemicalCAS HazardsEngineering Controls Needed for Usage and StorageDisposal
Trichloroacetic acid (TCA) (3%)Causes severe skin burns and eye damage. Very toxic to aquatic life with long lasting effects.FumehoodDispose in accordance with national/local regulations.
Protecting Group (DCC)Harmful if swallowed. Toxic in contact with skin. May cause an allergic skin reaction. Causes serious eye damage.FumehoodDispose in accordance with national/local regulations.
DichloromethaneCauses skin irritation. Causes serious eye irritation. May cause drowsiness or dizziness. Suspected of causing cancer.FumehoodDispose in accordance with national/local regulations.
Phosphoramidite monomerNASchlenk linesDispose in accordance with national/local regulations.
TetrazoleHighly flammable liquid and vapor. Harmful if swallowed, in contact with skin or if inhaled. Causes serious eye irritation.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
Acetic anhydrideFlammable liquid and vapor. Harmful if swallowed. Causes severe skin burns and eye damage. Fatal if inhaled.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
PyridineHighly flammable liquid and vapor.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
THFHighly flammable liquid and vapor. Harmful if swallowed. Causes serious eye irritation. May cause respiratory irritation. May cause drowsiness or dizziness. Suspected of causing cancer.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
N-methyl imidazoleCombustible liquid. Harmful if swallowed. Toxic in contact with skin. Causes severe skin burns and eye damage. Suspected of damaging fertility or the unborn child.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
AcetonitrileHighly flammable liquid and vapor. Harmful if swallowed, in contact with skin or if inhaled. Causes serious eye irritation.Fumehood, Flammable cabinetDispose in accordance with national/local regulations.
ArgonContains gas under pressure; may explode if heated.Gas tankContact licensed processional waste disposal service.
I2Harmful if swallowed, in contact with skin or if inhaled. Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation. Causes damage to organs (Thyroid) through prolonged or repeated exposure if swallowed. Very toxic to aquatic life.FumehoodDispose in accordance with national/local regulations.
Ammonia hydroxideCauses severe skin burns and eye damage. May cause respiratory irritation. Very toxic to aquatic life. Toxic to aquatic life with long lasting effects.FumehoodDispose in accordance with national/local regulations. Cannot be poured down drain.

Notably, many of these reagents require specialized training and engineering safety measures in place to procure and use.

Manufacturing, Processing, Formulation

image.png

Figure 1: Example of Automated Chemical DNA Synthesis Machinery2

Table 2: Materials used in Chemical Synthesis
MaterialDescription
Filterstandard 1/16 in X-tra fine high-density hydrophobic polyethylene sheet
TeflonUsed for chemical resistance
BottlesStorage for reagents
TubingConnects different parts of the apparatus
Solenoid ValvesControl flow of reagents
ManifoldDistributes reagents evenly
10 vesselsStorage of each solvent
Beads0.7 mg beads for 20 nmol

Steps (for 40 nmol of DNA)

  1. Beads washed three times with 110 ul ACN
  2. Dry with vacuum pulse
  3. Detritylation: 4 repetitions of 6 s exposure to 60 ul of TCA followed by four washes with ACN
  4. Dispense 25 ul of phosphoramidite bases, followed by tetrazole
  5. After 1 min, two washes with ACN
  6. 15 s exposure to 20 ul of iodine solution
  7. Beads given final series of five washes with ACN at end of cycle
  8. 0.5 ml 30% ammonium hydroxide, vials capped and heated for 1hr at 85C
  9. Vials are chilled, uncapped, oligomers are evaporated to dryness using 12 nozzle drying manifold

Enzymatic

The enzymatic synthesis of DNA strands relies on the production of TdT, which requires a series of protocols to successfully isolate it from E. coli. Some of these experiments, such as cloning, transformation, and protein isolation, are routinely performed in microbiology and synthetic biology labs. Figure 2 sequentially displays the steps and protocols used to isolate TdT. In contrast, Figure 3 illustrates the process of DNA strand elongation using the solid-phase platform and the TdT isolated in Figure 2.

INPUTS (1).png

Figure 2: Flowchart showing the system boundaries considered in the LCA of enzyme production and purification.

INPUTS (2).png

Figure 3: Flowchart showing the system boundaries considered in the LCA of solid phase synthesis.

The specific experiments, along with their inputs and outputs considered for this LCA, are detailed in the following document: List of experiments to account for in LCA

We compiled the total raw materials required for TdT production from the previously mentioned document and created a file detailing all the reagents used, including their quantities, manufacturing, distribution, and waste management characteristics. From this document, Table 3 summarizes the raw materials utilized from pre-inoculum to TdT purification, along with key information concerning its analysis under the LCA framework. Note that this table includes only the raw materials and residues from experiments that contributed to the development of our prototype; thus, we didn’t include the totality of all reagents used during the iGEM season.

Here is the file with the complete information of all the raw materials: Raw Materials for TdT enzyme Production at a Laboratory Scale

Table 3: Summary of complete table of stage 1. Pre-inoculum to TdT Purification
CategoryItemsEnvironmental Impact Overview
BiologicalDNA, E. coli cells, LB Broth, SOC media, GlycineDNA and cells have a low-to-moderate impact, requiring special handling and disposal methods (e.g., bleach, autoclaving)3,4.

GelsAgarose, Agar PowderEnergy-intensive purification processes. Both products are often imported from overseas (Japan), contributing to a higher transportation footprint. Typically autoclaved after use5,6.

Chemicals & ReagentsKanamycin, Solutions, Buffers, SDS, Acrylamide, Bis-acrylamide, Ammonium persulphate, TEMED, Comassie R250, Glacial acetic acid, Methanol, Dye, Tris base, IPTG, Imidazole, Potassium Phosphate, NaCl

These items often require energy-intensive chemical synthesis and are typically associated with moderate-to-high ecological footprints during manufacturing. Chemical transportation involves safety concerns, slightly increasing the overall footprint. Most require chemical waste disposal, with some chemicals being toxic to the environment or needing neutralization7,8,9,10.

Alcohols & SolventsGlycerol, MethanolBoth alcohols and solvents are produced through energy-intensive processes, with moderate ecological footprints. Their transportation also contributes to their overall impact, and need to be disposed of via chemical waste management11,12.

Salts & BuffersPotassium Phosphate, NaCl, Tris baseSimple salts have low manufacturing and transportation impacts. They can typically be disposed of down the drain, with minimal ecological consequences. However, more complex chemical buffers (e.g., Tris) require chemical waste disposal13.

UtilitiesAutoclave water and electricity consumption, energy use by machines (centrifuges, incubators, PCR machines)

Autoclaves and other lab machinery consume significant energy, adding to the overall footprint of the laboratory processes14, 15.

ResiduesPipette tips, gloves, PCR tubes, Eppendorf tubes, Falcon tubes, serological pipettes, and other lab disposable materials.

Plastic-based residues contribute significantly to the ecological footprint due to their production and non-recyclable nature. Items like gloves and pipette tips are typically discarded as biohazard waste16,17,18.

Similarly, Table 4 summarizes the raw materials used for SPS while considering their footprint. The same limitations apply as in Table 3.

Table 4: Summary of complete table of stage 2. Downstream (DNA strand synthesis—SPS)
CategoryItemsEnvironmental Impact Overview
BiologicalLB Broth, NeutravidinLB broth has a moderate ecological footprint due to biological extracts and salts, with autoclaving required for disposal19.

Chemicals & ReagentsKanamycin, IPTG, Bis-acrylamide Urea, TEMED, APS, Primer, dNTP, NaOH, Biotinylated Primer, Biotin-PEG-SVA, CoCl2, NaOH

These chemicals require complex synthesis processes, contributing to moderate-to-high ecological footprints. Their transportation, especially with chemical safety concerns, adds to the impact. Most require careful handling and disposal as hazardous or chemical waste7,20,21,8,3,22,23.

Dyes & IndicatorsDyeSmall amounts are used, leading to a relatively low manufacturing and transportation footprint. However, chemical waste disposal is still required24.

GasesNitrogen GasNitrogen is produced by fractional distillation and transported in pressurized containers, leading to a moderate ecological footprint, particularly in transportation25.

ResiduesPipette tips, PCR tubes, Falcon tubes (15 mL & 50 mL), Cuvettes, Kim wipes, Aluminum foil, Eppendorf tubes, Gloves

Most of these items are plastic-based and non-recyclable, contributing significantly to laboratory waste. Gloves, in particular, have a high environmental footprint due to toxic chemicals used in production, and are typically disposed of as biohazard waste16,26.

Life Cycle Analysis

Chemical

Because we do not have the resources to conduct our own analysis of chemical synthesis, we will use reported values for the 12 Principles of Green Chemistry, but highlight the some below that we want to compare to enzymatic synthesis 27:

  1. Prevention of Waste:
    1. Chemical synthesis produces large amounts of organic and aqueous waste, require specialized and energy intensive methods for disposal, contributing significantly to environmental pollution.
  2. Atom Economy: 36% for deoxynucleoside phosphramidies, indicating inefficiency in how raw materials are converted into final products.
  3. Less Hazardous Chemical Synthesis: Many reagents listed above are hazardous to human health, increasing safety risks for laboratory personnel, and if realized to the environment, can be detrimental to wildlife.
  4. Safer solvents and Auxiliaries: The reliance on solvents such as acetonitrile, dichloromethane (DCM), triethylamine, and pyridine poses risks due to their toxicity and flammability.
  5. Catalysis: absence of catalysts in chemical synthesis means reactions can be less efficient and require harsher conditions, further increasing environmental impact.

Other points that contribute to increased energy usage, difficulties in handling + storage and environmental pollution:

  1. Safety and Handling: Given the toxic and hazardous nature of the reagents, stringent safety protocols are necessary, including the need to store solvents separately and mix them only during synthesis. This increases operational complexity and risk.
  2. Reaction Environment: The need for specialized equipment (e.g., fume hoods, flammable cabinets) increases the overall energy footprint and cost of the synthesis process. Additionally, the release of vapours, although dilute, still contributes to environmental pollution
  3. Process Time and Complexity: The multi-step nature of chemical synthesis, which often requires extensive washing and purification, adds time and resources to the overall process.

image.png

Figure 4: Oligonucleotide synthesis cycle27.

A common metric to quantify waste byproducts, leftover reactants, solvent losses, spent catalysts and catalyst supports, is E-factor28. Reports mention that chemical synthesis E-factor is 4700 Kg of waste per 1 Kg of product29. Thus, there are many ways that chemical synthesis can stand to be improved, either through enzymatic means or innovations in chemistry.

Additionally, because of the toxicity/harzadous nature of the reagents being used, the apparatus above requires solvents to be stored separately and only mixed together during synthesis. We will see below that our wet lab team can create a buffer to be used in the reaction mixture, essentially a one-pot reaction for synthesizing DNA with TdT.

The points above will be compared with enzymatic synthesis, and how nuCloud can help tackle these pain points in DNA synthesis.

Enzymatic

Enzymatic synthesis benefits significantly that due to the enzymes’ nature, it doesn’t require as many chemicals to elongate a DNA strand compared to chemical synthesis. The only significant chemical used is NaOH, and even then, its quantities are in the microliter range. On the other hand, chemical synthesis uses phosphoramidite and involves complex chemical processes that require toxic and volatile reagents. Furthermore, enzymatic synthesis occurs in water and salt buffers, dramatically reducing the ecological footprint of the actual synthesis process30.

While the enzymatic synthesis is friendlier to the environment, it is also important to focus on the production and isolation of the enzymes doing the elongation. Using the experimental data we gathered throughout the iGEM season, we were able to quantify the amount of reagents used to produce ThTdT as well as the physical waste generated. We also considered some of the utilities involved in this process. The largest quantities of reagents used include ~1.6 L of deionized H2O, a total of 912.5 mL for solutions, and 600 mL of methanol. Other significant quantities are 200 mL of glacial acetic acid and 100 g of ammonium persulphate. Compared to chemical synthesis, the chemicals and reagents used in the enzyme production stage are less volatile and toxic.

However, the main environmental challenges arise from media used to grow the cells (950 mL of SOC), reagent transportation, and the total waste generated. Like in most laboratories, the amount of lab plastic waste is significant, around 1.695 Kg of plastic (LDPE, PVC, PP, PS, PE) in our case, and most of it cannot be reused due to contact with biohazardous substances. Moreover, to be properly disposed of, most waste must be autoclaved, which consumes significant energy and adds to the overall footprint of the laboratory processes involved. While chemical reagents, such as bleach, could be used as alternatives, this would make enzymatic synthesis dependent on toxic chemicals. That said, the disposal of organic solvents in chemical synthesis is much more environmentally taxing as it often requires inciniration or a specialized operation 31.

To address these challenges, we developed a microfluidic platform and bioreactor. The microfluidic platform reduces reaction time, improves sensitivity and control, and simplifies automation and parallelization. By precisely controlling reaction volumes, it allows us to use significantly fewer reagents, as the increased surface-to-volume ratio enhances mass transfer and accelerates reaction rates through more molecular collisions. The platform’s design also enables better control over reaction conditions, optimizing enzyme activity and increasing product detection sensitivity, thus reducing waste. Likewise, our bioreactor creates optimal growth conditions, leading to higher cell densities and greater enzyme yields, making the process more resource-effective, especially in large-scale applications.

It is also important to note that once the cells are initially transformed and start producing the plasmid of interest in our bioreactors, the process will become simpler, using fewer chemicals and generating less plastic waste. This will improve disposal efficiency and may open up recycling options during production.

In summary, by streamlining and scaling production with the microfluidic platform and bioreactor, we reduce reagent and plastic waste while improving the overall efficiency of TdT production and isolation. Learn more on our Hardware page.

image.png

Figure 5: Enzyme-catalyzed single base extension using TdT27.

Life Cycle Improvements

Chemical

Improvements in chemical synthesis of DNA include moving away from solvent-based methods towards mechanochemistry, by functionalization on an ionic liquid support29, to eventually a one-pot synthesis.

Of course, we can also move towards enzymatic methods, which typically require less toxic reagents, especially if biocatalysts are being used, and simpler procedures. In fact, one-pot synthesis is already archived with enzymatic synthesis, and by extension nuCloud.

Enzymatic

Improvements in our enzymatic synthesis platform could involve testing different protocols to optimize reaction conditions and reduce the use of toxic reagents in TdT production. Exploring alternatives to harmful chemicals like acrylamide and kanamycin may also be worthwhile, as these substances contribute to a higher carbon footprint due to their energy-intensive production. Shifting to safer alternatives could lower both environmental and health impacts. Additionally, optimizing equipment like autoclaves through batch processing or integrating alternative energy sources could further reduce energy consumption. Lastly, enhancing bioreactor designs for easier cell harvesting and purification would significantly cut resource demands and waste generated.

Limitations

This LCA-inspired page has several key limitations that need to be addressed. First, we were unable to quantify the electrical and water usage of lab equipment, such as autoclaves, bioreactors, and common devices like PCR machines, which is crucial for a clearer understanding of the process’s resource demands. Additionally, we didn’t establish a chemical synthesis control reaction to accurately measure reagent consumption and waste generation, which would have enabled a better comparison between the two in terms of plastic waste and reagent use. Another important factor we couldn’t include, due to lack of data, is the ecological footprint of transporting reagents. Finally, this page lacks a standardized unit for quantitative comparison between chemical and enzymatic methods. Using a common environmental footprint unit would have allowed for a more thorough evaluation instead of relying solely on qualitative assessments. For the next iteration of this document, the above mentioned will be included.

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