Week 1 HW: Principles and Practices

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Week 2 HW:DNA Read, Write & Edit

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Week 3 HW:Lab Automation

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Week 4 HW: Protein Design Part 1

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Week 5 HW: Protein Design Part 2

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Week 6 HW: Genetic Cirscuits Part 1

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Week 7 HW: Genetic Circuits Part 2

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Week 9 HW: Cell Free Systems

Homework question from Peter Nguyen

1.Write a one-sentence summary pitch sentence describing your concept. I could use cell free systems adapted to producing inaK in order to directly inoculate glaciers with the aim to preserve and boost ice formation, it would help glaciers rebuild and be more resistant to increasing temperatures caused by climate change. However, I would be interested in pushing the idea of geotextiles already helping preserve glaciers and design a living material, with inaK with a boosted ice nucleation function to create proactive glacier covering actively working to rebuild and preserve glacier ice.

2.How will the idea work, in more detail? Write 3-4 sentences or more. The inaK would be synthesized through a cell free model, using an alternative to E. coli which could resist and be active in sub-zero conditions (to stay active in glaciers), such as Oleispira antarctica a psychrophiles bacteria which has evolved to have specialized ribosomes and enzymes able to remain flexible and functional in a frozen environment. Oleispira antarctica contains unique chaperone (Cpn60 and Cpn10) preventing protein misfolds in frozen temperatures. This cell design would include pores in the membrane so it can stay alive in the textile by having an ATP source of input. These then freeze dried cells would be put into a textile (inoculated during the making of the textile), the textile can be brought to location and installed on the glacier and then be rehydrated to allow ice nucleation of the glacier to begin. This would permit me to create a live material that would be dormant in production and transportation and control its freezing function (preventing the textile from accidentally freezing its surrounding). Note that because I am working with ice nucleation there might be challenges in freeze drying these cells.

Reference List Ferrer, M. et al. (2003) ‘Low temperature-induced systems failure in Escherichia coli: Insights from rescue by cold-adapted chaperones’, Journal of Biological Chemistry. Cui, Y. et al. (2022) ‘Cell-free PURE system: evolution and achievements’, Biodesign Research. D’Amico, S. et al. (2006) ‘Psychrophilic microorganisms: challenges for life’, EMBO reports.

3.What societal challenge or market need will this address? This addresses the environmental, social and political issue of melting glaciers caused by climate change, which only increases the power of climate change as glaciers are key factors in slowing climate change. We are actively losing biodiversities and ecosystems and doing very little about it. It is not seen as a profitable income so little motivation is inputted. However, in the longterm, this irreversible damage done to our nature will actively make climate change worse, and there will be many destructive environmental, social and economic consequences driven by this overlooked issue.

4.How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? Working at very large scale, scale of the glacier, it can be a challenge to efficiently rehydrate the living material as it would be very energy consuming and costly to do it manually, but, if the living geotextile is strategically implemented at the right time of the year (early spring, already when they glacier coverings are usually installed) then nature itself through rain could activate the material naturally. The aim is to limit the human labor impact and simply give nature a tool to reinforce what it already knows how to do. Considering the one time use issue, geotextile coverings which are already used to protect glaciers are removed and installed yearly according to their natural ice melting and forming cycles. The next step of my research would be to find a way to keep the textile created and reabsorb it with new inaK cell free protein systems when it is needed next. The goal is to create a regenerative textile and closed loop system to avoid waste through one time solutions.

Homework question from Ally Huang

1.Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words) I am interested in exploring the purpose of ice nucleation in cell free design, freeze dried or not, taking the shape of a multipurpose textile which can be used as an alternative to current voluminous refrigeration tools or a freezing textile to activate. In space stations like the ISS a lot of research relies on lab samples being preserved in sub zero temperatures, from human research samples to organisms or protein crystals. Within research some experiments need cold induced phase changes to be activated or triggered. This technology could also be used for food or medical supplies.

2.Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words) The genetic target of this project would be the inaK ice nucleating protein, commonly found in Pseudomonas syringae, with a wide potential of freezing functions.

3.Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) The challenge is optimizing cold packaging and storage systems, a material which would require less space or a material able to be activated once in space again having less constraints in terms of space while travelling. The inaK offers a variety of possibilities in a cell free system whether freeze dried or not as it has a focused and controlled function to freeze. According to the development of the product it can be chosen at what temperature it freezes or activates and how resistant it can be to external temperatures. Creating a highly controlled and bespoke design for certain use in space allows for better control on the research done in space, every aspect of the research can be tailored in hopes to improve success rates of experiments. InaK is a relatively easy INP to work with.

4.Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) I am interested in creating polyvalent designs with multiple usages and applications, this project aims to find an optimal alternative refrigerating system which can have bespoke qualities specific to in space research. As small of a detail it might seem every aspect and tool of experiments impacts the result of research and can lead to better efficiency, results or unexpected breakthroughs. During a space mission all equipment has to be optimized due to lack of space and need for many items and a polyvalent tool that can respond to a wide range of uses can help with the space optimization.

5.Outline your experimental plan - identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words) I would design a cell free system for the inaK ice nucleating protein, freeze dry some and then create living textiles, some active and some dormant. The practicality of a textile is that it can be molded, cut, sewn, layered to adapt to any existing object which would then need a freezing function. I can control the amount of inaK for the freezing rate needed, experiment with the different temperatures it can freeze at and the different temperatures it can stay frozen at, I can explore the threshold of the inaK. I would then test the reactivation rates, how much water is needed and how long it would take.

Week 10 HW: Advanced imaging & measurement technology

HTGAA Week 10 Advanced imaging & measurement technology

Final Project

1.Please identify at least one (ideally many) aspect(s) of your project that you will measure. It could be the mass or sequence of a protein, the presence, absence, or quantity of a biomarker, etc.

As for this project I aim to use inaK for ice production I would like to measure the ice nucleation ratio and efficiency of the inaK protein. Additionally, I would like to measure the temperatures inaK can resist to, on its own and as a supplement to an ice sample. If my initial experiments are successful I would like to measure the inaK ratio innoculated into ice to find the most optimal inaK quantity needed.

2.Please describe all of the elements you would like to measure, and furthermore describe how you will perform these measurements.

To measure the ice production ratio and efficiency of inaK I could use differential scanning calorimetry (DSC) which measures the difference in the amount of heat required to increase the temperature in the sample compared to a reference. It measures the ice nucleation ratio by calculating the enthalpy which is the area below the peak, allowing me to understand precisely how much of the water in the cell is being converted to ice. It measures the efficiency of ice nucleation by creating an exothermic peak ( release of a burst of energy) and analysing how high of a temperature the inaK can still function. This should give me information on thermodynamic efficiency. I can also measure nucleation temperature through a droplet freezing assay for smaller samples allowing me to test a multitude of potential solution mixes. Here a high speed camera paired with a cooling plate ( a Linkam for example) can allow me to assess how fast a droplet of a solution containing inaK can freeze. Testing this on multiple samples containing different amounts of inaK will give me a spectrum of freezing capacity to find the most optimal ratio of inaK. This experiment could be coupled with an infrared thermography technology which will capture the heat spike and nucleation rate of an inaK and understand how fast the ice nucleation spreads through the cell membrane.

Reference List Schmid, D. et al. (2016) ‘A high-throughput assay for the characterization of ice-nucleating proteins’, Biophysical Journal. This study outlines the specific use of droplet assays to quantify InaK efficiency.

3.What are the technologies you will use (e.g., gel electrophoresis, DNA sequencing, mass spectrometry, etc.)? Describe in detail.

For these experiments I will use a Differential Scanning Calorimeter for the DSC, a high precision camera, a cooling plate and IR thermography.

Waters Part 1 - Molecular Weight

For this section I used a combination of the tools provided, my knowledge and AI assistance as I have trouble understanding math related work.

1. Based on the predicted amino acid sequence of eGFP and any known modifications, what is the calculated molecular weight ?

According to Expasy I found that this sequence has a theoretical pI/Mw of 5.90 / 28006.60. To calculate the molecular weight of this sequence I referred to the standard isotopic mass of amino acids and subtracting H²O for each peptide bond. For this amino acid sequence I found that : -eGFP of 238 AA = 26.735.6 Da -LE Linker of 2 AA = 242.3 Da -x-His Tag of 6 AA = 822.8 Da Resulting in a total molecular weight of 27800.7 Da

2. Calculate the molecular weight of the eGFP using the adjacent charge state approach described in the recitation. Select two charge states from the intact LC-MS data (figure 1) and:

MW : molecular weight in Daltons m/z : value of the peak on the x-axis of the spectrum z : integer charge state of said peak H+ : mass of a proton

a.Determine z for each adjacent pair of peaks $(n, n+1)$ using: $$ {\large z} = {\Large \frac{\frac{m}{z_{n+1}}}{\frac{m}{z_n} - \frac{m}{z_{n+1}}}} $$

Here I am using the adjacent peaks at 875.4421 and 903.7148. m:zn = 875.4421 m:zn+1 = 903.7148 The lower the m/z the higher the charge versus the higher the m/z the lower the charge. Using the provided formula z = 903.7148 : (903.7148 – 875.4421) = 903.7148 : 28.2727 = 31.96 Charge states must be integers and the charge state for the peak at 903.7148 is z= 31 So, the peak at 875.4421 has a charge state of z+1=32

b.Determine the MW of the protein using the relationship between $\frac{m}{z_n}$, $MW$, and $z$

The base equation for the peaks in this figure is m:z = (MW + (z x H+)) : z Here I rearrange the formula to find MW MW = z x (m:z) - (z x H+) If z=31, then, MW = 31 x(903.7148) - (31x1.008) MW = 28 015.1588 – 31.248 MW = 27 983.91 Da

c.Calculate the accuracy of the measurement using the deconvoluted MW from 2.2 and the predicted weight of the protein from 2.1 using: $$ \text{Accuracy} = \frac{|MW_{\text{experiment}} - MW_{\text{theory}}|}{MW_{\text{theory}}} $$

Using the provided formula and substituting the values accordingly, If, Accuracy = (MW experimental - MWtheory) : MW theory Then, Accuracy = (27 982.90 - 27 782.70) : 27 782.70 Accuracy = 200.20 : 27 782.70 Accuracy = 0.0072 =0.72%

3.Can you observe the charge state for the zoomed-in peak in the mass spectrum for the intact eGFP? If yes, what is it? If no, why not?

One can observe the charge state in the zoomed in peak but not using the previous calculation method using the adjacent peak method used for the entire spectrum. The zoomed in area presents an isotopic cluster of one charge state, therefore, the observation is focused on isotopic resolution. The other peaks represent different amounts of protons. So, here, yes I can see the charge state using isotopic resolution because the peaks are distinct and separate and the instrument has high enough of a resolution to expose the isotopes separately. It is because the instrument used here is of high precision that we are able to have high resolution, if a lesser precise instrument were used then it would be very difficult to see the charge state. The formula to calculate isotopic spacing here would be z = 1 : Δm/z

Waters Part 2 - secondary & tertiary structure

1.Please explain the difference between native and denatured protein conformations. For example, what happens when a protein unfolds? How is that determined with a mass spectrometer? What changes do you see in the mass spectrum between the native and denatured protein analyses (figure 2)?

There is a structural difference between the native and the denatured protein conformations, the native protein conformation is a tightly folded protein, a unique three dimensional structure unique to its biological environment. The structure is compact but is held by weak non-covalent bonds. In contrast, a denatured protein conformation is the unfolded structure once the weak bonds are broken, the structure is flexible and unpredictable resulting in a random coil shape. The denatured protein no longer has a function compared to the native protein conformation. A mass spectrometer scans the protein shape by measuring its mass and charge rather than measuring the shape directly. The top green graph corresponds to the denatured confirmation and the bottom red chart corresponds to the native conformation. The denatured conformation graph shows crowded peaks in the 500 to 1200 m/z range, these are low m/z values. A lower m/z equals a higher z charge and when a protein is unfolded into a random coil then the basic residues are exposed to a proton rich environment and can easily be protonated creating a protein with a very high net positive charge. The native conformation has larger m/z values with peaks at 2545 and 2799, the higher the m/z value the lower the z charge. When the protein is still tightly folded the basic residues are buried in the hydrophobic core protecting it from its environment meaning protons cannot impact them so the protein is less protonated.

2.Zooming into the native mass spectrum of the eGFP from the Waters Xevo G3 QTof or MS (figure 3), can you discern the charge state of the peak at ~2800? What is the charge state? How can you tell?

It is possible to discern the charge state of the peak at ~2800 m/z because instrument provides a high resolution even if the image focuses on the 2525 m/z peak, we are still able to see the isotopic distribution by measuring the distance between the individual isotopes and we would be able to calculate the charge state. Measuring the charge state of the peak at ~2800 m/z using isotopic spacing, In a mass spectrum the individual isotopes of the same molecule differs by approximately 1 Dalton, The distance between the isotopes here on the x-axis Δm/z can be calculated using the following formula, Δm/z = 1: z Zooming in at the 2799.4199 peak same as for the 2545 peak (with a peak separation of precisely 0.1 m/z) then, z = 1: 0.1 = 10 So the charge state of the peak at ~2800 m/z is +10

Waters Part 3 - Peptide mapping, primary structure

1.How many Lysines (K) and Arginines (R) are in eGFP? Please circle or highlight them in the eGFP sequence given in Waters Part I question 1 above. (Note: adding the sequence to Benchling as an amino acid file and clicking the biochemical properties tab will show you a count for each amino acid).

2.How many peptides will be generated from tryptic digestion of eGFP?

I found 19 peptides were generated from this sequence using the trypsin.

3.Based on the LC-MS data for the Peptide Map data generated in the lab ( please use Figure 5a as a reference) how many chromatographic peaks do you see in the eGFP peptide map between 0.5 and 6 minutes? You may count all peaks that are＞10% relative abundance.

Between 0.5 and 6 minutes there are 14 distinct peaks above 10% relative abundance in this figure.

4.Assuming all the peaks are peptides, does the number of peaks match the number of peptides predicted from question 2 above? Are there more peaks in the chromatogram or fewer?

In comparison to the amount of predicted peptides, 19, there are fewer peptides in the chromatogram.

5.Identify the mass-to-charge (m:z) of the peptide shown in Figure 5b. What is the charge (z) of the most abundant charge state of the peptide (use the separation of the isotopes to determine the charge state). Calculate the mass of the singly charged form of the peptide ([M+H]+) based on its m:z and z.

The z charge of the most abundant peak in this peptide is m/z = 525.76712 To calculate the charge state, First isotope peak 525.76712 Second isotope peak 526.25918 Calculating the spacing, (Δm/z):526.25918 - 525.76712 = 0.49206 Using the formula, z = 1: 0.49206≈2.03 So the charge state z of the most abundant peak of the peptide is of +2

Calculating the mass of the singly charged form of the peptide ([M+H]+), First I need to calculate the neutral mass M using M=zx(m/z)-(zx1.00727), 1.00727 Da is the mass of a proton, So, M = 2 x (525.76712) - (2x1.00727) M = 1051.53424 - 2.01454 = 1049.5197 Da ([M+H]+) can be calculated by adding one proton mass back to the neutral mass, [M+H]+ = 1049.5197 + 1.00727 = 1050.5270 Da

6.Identify the peptide based on comparison to expected masses in the PeptideMass tool. What is mass accuracy of measurement? Please calculate the error in ppm. (Recall that Accuracy = (MW experimental - MWtheory) : MW theory)

To identify the peptide I consider that the experimental neutral molecular sight (MWexperimental) was calculated for the peak 525.76712 m/z and equaled MW: 1049.5197 Da, I will use the following peptide sequence as a theoretical tryptic digest as a comparison, LPDNHYLSTQSALSK, and considering theoretical MW (MWtheory) : 1049.5393 Da. This peptide corresponds to the residues 139–153 of the eGFP protein.

To calculate the mass accuracy, error in ppm (parts per million) I will use the following formula, Accuracy (ppm) = ((MWexperimental - MW theory) : MWtheory) x 106 Now adding the values, MW experimental = 1049.5197 MW theory = 1049.5393 Accuracy (ppm) = ((1049.5197 - 1049.5393) : 1049.5393) x 106 Accuracy (ppm) = (-0.0196) : 1049.5393) x 106 Accuracy (ppm) = –18.67ppm

7.What is the percentage of the sequence that is confirmed by peptide mapping ? (see figure 6)

The percentage of the sequence that is confirmed by peptide mapping seems to be indicated at 88%, the blue highlighted areas are confirmed amino acids in the sequence.

Waters Part 4 - Oligomers

We will determine Keyhole Limpet Hemocyanin (KLH)’s oligomeric states using charge detection mass spectrometry (CDMS). CDMS single-particle measurements of KLH allow us to make direct mass measurements to determine what oligomeric states (that is, how many protein subunits combine) are present in solution. Using the known masses of the polypeptide subunits (Table 1) for KLH, identify where the following oligomeric species are on the spectrum shown below from the CDMS (Figure 7): -7FU Decamer -8FU Didecamer -8FU 3-Decamer -8FU 4-Decamer

I will first calculate the theoretical mass for the species using the given measures 7FU = 340 kDa and 8FU = 400 kDa. The axis of the spectrum is in MDa (megadaltons) where 1MDa = 1000 kDa.

I can then identify the peaks on the spectrum -7FU Decamer ≈ 3.40 MDa is the peak labeled 3.4 on the spectrum -8FU Didecamer ≈ 8.00 MDa correspond to the peak labeled 8.33 -8FU 3-Decamer ≈ 12.00 MDa matches the peak labeled 12.67 -8FU 4-Decamer ≈ 16.00 MDa would appear in the cluster of small unlabeled peaks around 16 to 17 MDa on the far right side of the chart

Waters part 5 - Did I make GFP?

1. Please fill out this table with the data you acquired from the lab work done at the Waters Immerse Lab in Cambridge, or else the data screenshots in this document if you were unable to have lab work done at Waters.

Week 11 HW: Building genomes

Week 11 Bioproduction & Cloud Lab

Part A - The 1.536 pixel art work canvas, collective artwork

1.Contribute at least one pixel to the global artwork

I added early on a pixel towards the top left corner. I do not have much to say about this section of the work except maybe understanding the full purpose of this exercise.

Part B - Cell Free protein synthesis, cell free reagents

1. Referencing the cell-free protein synthesis reaction composition (the middle box outlined in yellow on the image above, also listed below), provide a 1-2 sentence description of what each component’s role is in the cell-free reaction.

E. coli Lysate

• BL21 (DE3) Star Lysate (includes T7 RNA Polymerase) : offers the base molecular machinery such as ribosomes, tRNAs and enzymes for translation, the Star Lysate strain reduces mRNA degradation, and, the T7 Polymerase drives high level transcription from T7 promoters

Salts / Buffer

• Potassium Glutamate : primary salt that maintains ionic strength and provides potassium ions essential to ribosomal function and protein to nucleic acid exchange
• HEPES-KOH pH 7.5 : chemical buffer which helps maintain a stable physiological pH which affects enzymatic function of the transcription and translation machinery
• Magnesium Glutamate : magnesium ions are vital contributors to stabilizing the ribosome structure and enabling catalytic activity of the polymerases kinases
• Potassium phosphate, monobasic and dibasic : functions as a secondary pH buffer and a source of inorganic phosphate essential for the regeneration of high energy molecules such as ATP

Energy / Nucleotide system

• Ribose : serve as a carbon backbone precursor for the synthesis of nucleotides, allowing for regeneration of NTPs essential for transcription and energy transfer
• Glucose : primary metabolic energy source fueled through glycolysis allowing to regenerate the ATP and GTP essential to the good functioning of protein synthesis
• AMP / CMP / UMP : offers nucleotide building blocks for RNA synthesis and can be converted into triphosphate such as ATP, CTP, UTP needed in transcription
• GMP : from the lack of GMP might demonstrate a dependency on salvage pathways to generate GTP essential to translation
• Guanine : precursor for GMP/GTP synthesis through salvage pathways helpful to RNA synthesis and ribosomal function Translation Mix (Amino acids)
• 17 Amino Acid Mix : provide the base building blocks to synthesize the polypeptide chain
• Tyrosine : supplied separately because of its solubility limitations, becomes an essential building block for protein synthesis once it is adapted into a usable form
• Cysteine : added separately due to its oxidation limitations, it is an essential compound in forming disulfide bonds in proteins Additives
• Nicotinamide : serves as a precursor for NAD+ / NADH synthesis reinforcing redox balance and metabolic reactions occurring in energy regeneration Backfill
• Nuclease Free Water : is used to adjust all the components to the desired the final reaction volume while it avoids degradation of DNA / RNA by nuclease and ensures stable transcription and translation processes

3. Describe the main differences between the 1-hour optimized PEP-NTP master mix and the 20-hour NMP-Ribose-Glucose master mix shown in the slide.

The main difference between the two master mix results in found in the energy and nucleotide sourcing a the 1-hour mix makes use of the PEP and pre-synthesized NTPs for instant and high burst protein synthesis compared to the 20-hour mix uses the ribose, glucose and NMPs as precursors to regenerate energy and nucleotides throughout time. Therefore, the 1-hour mix is designed for speed and rapid prototyping in contrast to the 20-hour mix allows to better optimize the cost for effectiveness by using the Lysate’s metabolic pathway to support the reaction for an extended period of time.

Part C - Planning the global experiment, cell-free master mix design

1.Given the 6 fluorescent proteins we used for our collaborative painting, identify and explain at least one biophysical or functional property of each protein that affects expression or readout in cell-free systems. (Hint: options include maturation time, acid sensitivity, folding, oxygen dependence, etc) (1-2 sentences each)

• sfGFP : provides robust and rapid protein folding therefore the protein is less likely to aggregate enabling it to offer a strong fluorescent readout even if fused to complex proteins
• mRFP1 : is a protein with a longer maturation time signifying the fluorescence develops slower after translation and might have a delayed signaling time in shorter experiments, it has a low acidity tolerance
• mKO2 : is fast maturing and has a relative acidity tolerance, meaning the fluorescence will be less visible in a lower pH context but the fluorescence could increase in longer cell free reactions
• mTurqoise2 : is a cyan protein with for a high quantum yield and high photostability making the fluorescence outread a great signal no matter the length of the reaction, it is very sensitive to pH
• mScarlet_I : engineered for fast maturing and high brightness allowing for stronger fluorescence signals compared to older red proteins
• Electra2 : protein engineered for very fast maturation making it very useful for fast consuming energy systems in an experiment where a rapid output is needed before the mix’s energy is used up

2.Create a hypothesis for how adjusting one or more reagents in the cell-free mastermix could improve a specific biophysical or functional property you identified above, in order to maximize fluorescence over a 36-hour incubation. Clearly state the protein, the reagent(s), and the expected effect. Could the mTurquoise2 yield be increased or accelerated through pH stabilisation. The reagents HEPES-KOH would be increased to 100mM and Potassium Phosphate would be increased to 15mM. This adjustment should increase the capacity of the buffer within the mater mix and should neutralize organic acid by products such as lactate and acetate generated during the 36-hour metabolism of glucose and ribosome. Because mTurquoise2 is very reactive to pH, preserving the pH environment at 7.5 would prevent the typically occurring rapid cooling of the cyan signal which usually occurs as the mix acidifies over time. Therefore, the high quantum of yield of mTurquoise2 is complete and optimized leading to a bright and stable cyan readout which won’t dim as the energy levels decrease.

3.The second phase of this lab will be to define the precise reagent concentrations for your cell-free experiment. You will be assigned artwork wells with specific fluorescent proteins and receive an email with instructions this week (by April 24). You can begin composing master mix compositions here.

4. The final phase of this lab will be analyzing the fluorescence data we collect to determine whether we can draw any conclusions about favorable reagent compositions for our fluorescent proteins. This will be due a week after the data is returned (date TBD!). The reaction composition for each well will be as follows:

• 6 μL of Lysate
• 10 μL of 2X Optimized Master Mix from above
• 2 μL of assigned fluorescent protein DNA template
• 2 μL of your custom reagent supplements Total : 20 μL reaction

Week 1 Lab: Pipetting

Individual Final Project

iKe

Abstract

Mountain glaciers are melting progressively due to climate change and human activity. I am inspired by the glaciers of the Italian Alps where my family is from and throughout generations have seen firsthand the glaciers progressively disappear. Glaciers are vital ecosystems which contribute to protecting nature and human existence.

I aim to use my knowledge in textiles, biology and biodesign to help preserve and rebuild glaciers using ice nucleating proteins (INPs).

I theorise that inoculating glaciers with modified INPs using cell free synthesis would help improve ice catalyzation and make glacier ice more resilient to face increasing temperatures. This method will work as a defensive tool helping restore natural and healthy glacier cycles benefiting a wider ecosystem and battling climate change.

Project overview

Growing up I spent a lot of time in the Italian Alps where part of my family originates from, within only 23 years of life I have seen first hand the glaciers of mountains surrounding me disappear progressively, my mother sees an even bigger decrease and my grandfather a shocking decrease. With my background in textiles, my current studies in Biodesign, my curiosity for biology and now partaking in HTGAAA I will conceptualise a project combining textiles and biology as a means to create a tool which could help prevent the fast disappearance of glaciers.

Glaciers are vital elements to regulating the earth’s temperature, they are ecosystems of their own across the world. As pointed out by Glacier Preservation, “glaciers are essential for sustaining millions of people by providing fresh water, supporting hydropower generation, and playing a key role in environmental stability. However, as climate change accelerates, glaciers are retreating at an unprecedented rate, threatening water security, energy infrastructure, and increasing natural hazards like flooding and avalanches.” (Un-glaciers.org. (2026). Glacier Preservation is the Key to Ensuring the Security of Water, Energy, and Environmental Resources. [online] Available at: https://www.un-glaciers.org/en/articles/glacier-preservation-key-ensuring-security-water-energy-and-environmental-resources.).

My project aims to ethically preserve glaciers and help them naturally rebuild while respecting their natural cycles and having minimal interference. I want to work with ice nucleating proteins, specifically the inaK strands, they are commonly found in nature and have the function to catalyze ice. These INPs should be used to improve ice formation in glaciers and sustain ice levels while they face a rise in temperatures. I aim to work using a cell free synthesis method, potentially improve the inaK function through gene mutation and through different levels of test innoculate glaciers with inaK.

Aim 1 synthesize inaK ice nucleating protein (INP)

• Using UniProt source records (O30611) the ice nucleating protein DNA sequence of inaK (found in Pseudomonas Syringae - pathogenic)
• Refine DNA sequence through codon optimization using Twist
• Using Benchling and Twist create a plasmid for the inaK INP sequence
• Attempt cell free protein synthesis for inaK INP. This method provides more stability and less toxicity. Using one of the following methods
  • Detergent process
  • Liposome process
  • Nanodisc process

Aim 2 Further development of INPs in glaciers

• Consider genetically modifying inaK in order to increase ice catalyzation capacities.
• Use peptide motifs to improve the ice catalyzation function, enhancing or stabilizing the ice binding surface through crystal lattice. INPs like inaK a large and repetitive proteins, using peptide motifs allows to focus the INP.
  • Tandem N-terminal repeats: improve sequence stability enabling to boost ice nucleation
  • Aline rich helical peptides: freezing at higher temperatures due to aggregation of high concentration of aline rich peptides
  • TxT motif : uses hydroxyl to anchor water
  • SLT motif : stabilizes the organisation of water interface
  • Y motif: helps align water molecules

Could this method boost ice formation further without interrupting the natural cycle of glacier ice melting and rebuilding?

Aim 3 Future INP output reinforcing glacier ice formation

• Introduce successfully synthesized protein into a living material inspired by existing geotextile
• Observe if protein is able to stay alive. Observe how it may catalyse ice independantly
• Place living material over a sample of ice and observe the effect of the INP on ice. Is there an increase in ice formation?
• Hopefully obtain sample of a glacier and test the success rate of glacier inoculation of inaK.
• Introduce technology to glaciers
• If this technology allows glaciers to rebuild stronger and be less prone to melting this would have a significant positive impact on reversing climate change

Note that if CFPS fails, then attempt in vivo synthesis and introduce inaK to new host, Pseudomonas Fluorescens, which does not contain any ice-nucleotides, already exist within glaciers and presents no risk to nature. Transcribing new DNA data from one Pseudomonas to another will be either as they are part of the same species. Additionally, because Pseudomonas Fluorescens already exist within glaciers it facilitates horizontal gene transfer but also avoids disrupting the natural balance and ecosystem of the glaciers.

Within this project I will also focus a section of my research in ethics, analyzing the impact my work could have on glaciers and their ecosystems. I aim to be educated about environmental laws and bioethics.

I recognise that within the context of this project I would need the expert knowledge of a glaciologue able to provide me with specific and reliable data to use within my research which would allow me to work as ethically, sustainably and durably as possible.

Literature review

1. Existing methods for glacier preservation

Start by reviewing current glacier-preservation approaches and their limitations. This gives the project a real-world anchor and shows why controlling ice nucleation could matter. There are a few ongoing projects which aim to protect glaciers. Mainly they appear in the form of geotextile covering. GlacierProtect by Naue is a glacier protection project through geotextiles, it uses sustainable raw materials to reflect up to 75% of sunlight. Glaciers already have the capacity to reflect sunlight but this new textile supports this natural ability which preserves the glaciers and by preserving glaciers will enable them better form and themselves better reflect the sunlight. This project inspires me as a way of demonstrating these innovative projects are scalable but also that true regenerative design can be achieved with a mindset of supporting glaciers to promote their rebuild and growth where in turn they could thrive and function as they were meant to. Ponte di Legno Tonale glacier project with a similar technology to Naue, the Presena glacier has been protected since 2008 after observing significant damage to the glacier in 2000 by “covering Presena glacier with geotextile fabric covers, which are able to reduce by 50% the melting of snow and ice during summer months” (Ponte di Legno Tonale, n.d.). This project demonstrating chronological progress shows the real potential and benefit of protecting glaciers and assisting them to rebuild naturally.

Furthermore, many companies are innovating and making use of ice nucleating proteins in snow canons to increase production of snow. Snowmax International has made great progress in this direction by using INP to make water free at higher temperatures because of a higher concentration of nuclides, increase ice catalyzation speed as it attaches to nuclides, and be more resistant in time as it makes use of less water therefore reducing evaporation capacity and due to its smaller crystal structures from stronger iconic bonds. According to GoldBio, “P. syringae’s ice-nucleating activities have long been used to make artificial snow. Products including Snomax® use the proteins derived from the outside of bacteria to enhance the snow generated by snow blowers. One study showed that Snomax® increases the amount of snow made by a snow blower by as much as 90% (Snomax® International, 2013)” (Christner et al., 2008) leading to my next point of research. While this is mostly destined to ski track upkeep and not oriented to ice production it demonstrates that INPs are being used on a larger scale and prove to be effective. These technologies are also proving that they have no negative repercussions on the environment as they make use of all natural compounds.

Pseudomonas syringae is a bacteria which contains ice nucleating protein strands which have the ability to catalyze the formation of ice in sub zero temperatures. The main issue with pseudomonas syringae is that it is a pathogen in agriculture as it infects crops. However, would it be possible to extract the ice nucleating protein strands from the bacteria, use pseudomonas syringae as a model to synthesize ice nucleating protein or genetically modify the bacteria to neutralize its harmful effect to plants in order to use this genetically modified or synthesised bacteria as an ice forming agent to help preserve glaciers.

Instant ice packs are innovations of modern medicine where a sealed pouch of water is activated through an endothermic chemical reaction transforming surrounding heat into ice. Would it be possible to upscale this chemical reaction or let it inspire some biomimicry which through textiles and biology could use the increasingly hot temperature of the earth to transform it into ice on the surface of glaciers to help preserve them.

Snow Seeds by Tanay Wadokar is a project presented by a 2025 graduate of the MA Materials Future at Central Saint Martins (UAL, London). Tanay Wadokar created snow board stickers with cloud seeding technology. This would allow the deposit of ice nuclei while snowboarding which would enable locals and tourists to enjoy the inter sport while preserving mountains and glaciers by regenerating snowfalls. It is not about limiting the life we know today but rather by shaping it in a sustainable proactive way.

Reference list

Arnold, D.L. and Preston, G.M. (2019). Pseudomonas syringae: enterprising epiphyte and stealthy parasite. Microbiology, 165(3), pp.251–253. doi:https://doi.org/10.1099/mic.0.000715.

Boztas, S. (2024). Pumped up: will a Dutch startup’s plan to restore Arctic sea-ice work? The Guardian. [online] 27 Feb. Available at: https://www.theguardian.com/environment/2024/feb/27/climate-crisis-arctic-ecosystems-environment-startup-plan-pump-restore-melting-sea-ice-caps.

Christner, B.C., Morris, C.E., Foreman, C.M., Cai, R. and Sands, D.C. (2008). Ubiquity of Biological Ice Nucleators in Snowfall. Science, 319(5867), pp.1214–1214. doi:https://doi.org/10.1126/science.1149757.

Dr. Tobias Weidner, Dr. Janine Fröhlich-Nowoisky (2016). The effect of bacterial ice nuclei. [online] Www.mpg.de. Available at: https://www.mpg.de/10470442/ice-formation-bacteria-syringae.

James Dalton, Global Head, Water and Wetlands Team, IUCN (2025). Protecting glaciers – our most effective natural water manager. [online] IUCN. Available at: https://iucn.org/blog/202503/protecting-glaciers-our-most-effective-natural-water-manager.

McDonough, F. (n.d.). What is Cloud Seeding? [online] Desert Research Institute. Available at: https://www.dri.edu/cloud-seeding-program/what-is-cloud-seeding/.

Ponte di Legno Tonale. (n.d.). The protection of Presena Glacier. [online] Available at: https://www.pontedilegnotonale.com/en/pontedilegno-tonale-what-to-see/the-protection-of-presena-glacier/.

Roeters, S.J., Golbek, T.W., Bregnhøj, M., Drace, T., Alamdari, S., Roseboom, W., Kramer, G., Šantl-Temkiv, T., Finster, K., Pfaendtner, J., Woutersen, S., Boesen, T. and Weidner, T. (2021). Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water. Nature Communications, [online] 12(1), p.1183. doi:https://doi.org/10.1038/s41467-021-21349-3.

Snomax.com. (2015). FAQ - Snomax. [online] Available at: https://www.snomax.com/faq.html.

Steroplast Healthcare (2022). How Do Instant Ice Packs Work? [online] www.steroplast.co.uk. Available at: https://www.steroplast.co.uk/knowledge-base/how-do-ice-packs-work.html.

Un-glaciers.org. (2026). Glacier Preservation is the Key to Ensuring the Security of Water, Energy, and Environmental Resources. [online] Available at: https://www.un-glaciers.org/en/articles/glacier-preservation-key-ensuring-security-water-energy-and-environmental-resources.

Wadokar, T. (2025). Snow Seeds - Tanay Wadodkar - UAL Showcase. [online] Arts.ac.uk. Available at: https://ualshowcase.arts.ac.uk/project/635735/cover [Accessed 9 Feb. 2026].

Wellpott, V. and Wellpott, V. (2025). Glacier protection with geotextiles – A sustainable solution for the future. [online] Naue - Geosynthetics | Digtal Engineering Software | Installation services. Available at: https://www.naue.com/glacier-protection-with-geotextiles-a-sustainable-solution-for-the-future/.

2. Fundamentals of ice nucleation Explain the basic mechanism of ice nucleation, including:

• homogeneous vs heterogeneous nucleation
• the temperatures typically required
• why nucleation at higher subzero temperatures is valuable

Ice nucleation is a mechanism where water molecules transform from a liquid state to a solid crystalline lattice, an existing nucleus or template needs to surpass the energy barrier of crystallization.

In a pure water sample, containing no foreign particles, water molecules can spontaneously collide and form stable crystals, this is homogeneous nucleation. In contrast a heterogeneous nucleation is when a foreign compound, an ice nucleator, forms a physical surface or template for the water molecules to attach themselves to which significantly lowers the energy needed for freezing. In the case of Pseudomonas syringae the INPs provide a heterogeneous surface for ice to form itself on. Ice nucleation temperature will depend on the context in which ice nucleation is occurring, typically for pure water samples can stay liquid till -40°C in a supercooled state before freezing. With common nucleators such as minerals the water sample can freeze at about -10°C to -15°C. In the case of the inaK INP which I am studying, the ice catalyzation can be triggered at -2°C to -5°C, demonstrating the potential of using inaK compared to other INP. Being able to catalyze ice at higher subzero temperatures shows potential for environmental protection, as seen in the Snowmax project, helping to produce snow at higher temperatures, essential for protecting glaciers and reducing the ice loss. Additionally, they can have an atmospheric impact, because INPs are so effective they can truly affect weather patterns, similarly to cloud seeding technology they can trigger cloud formation and influence precipitation at warmer altitudes. For companies such as Snowmax the ice nucleation technologies allow them to produce the same amount of snow using less energy than they would need at regular ice nucleation temperature of -10°C or -15°C as machines don’t need to compensate for the temperature.

Reference List

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 25 April 2026).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15). doi: 10.1021/acs.jpcc.4c07422.

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 25 April 2026).

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

Experimental Data Figure (2026) Intact Mass Spectra: Native vs. Denatured sfGFP Analysis.

Experimental Data Figure (2026) Sequence Coverage Report: 88% Confirmation of sfGFP.

Experimental Data Figure (2026) CDMS Spectrum of KLH Oligomeric States.

3. Types of ice-nucleating particles Summarize the major categories of ice-nucleating particles:

• mineral
• biological
• organic/macromolecular
• engineered materials Then position biological ice nucleators as the most relevant class for your work.

Different types of ice nucleation particles exist, they can be organized in categories.

The mineral particles which are mostly inorganic atmospheric aerosols like minerals and soot. They are the most common INPs in the atmosphere but are not considered very efficient as they require the very low temperatures of -10°C to -15°C to initiate heterogeneous nucleation.

The biological ice nucleators which consist of bacteria such as the Pseudomonas syringae containing inaK. They are the most effective ice nucleators as they can trigger ice at higher temperatures, between -2°C and -5°C. Like the inaK protein they are usually very efficient due to their repetitive genetic code improving crystal lattice capability.

Organic and macromolecular compounds which refer to non-living organic substances. Polyols are an example of macromolecular compounds which are not always ice nucleators but can improve the efficiency of existing biological nucleators by up to 100 fold.

Engineered materials, however, are synthetic substances designed to imitate natural ice nucleation. Silver iodide for instance is a synthetic protein used in cloud seeding.

Biological nucleators such as inaK are the most relevant for my work as, on one hand, they prove to be more efficient for ice nucleation at higher sub zero temperatures which will be essential when working on glaciers confronted with rising temperatures. On the other hand, they have a better chance of non disruptive inoculation into glaciers as they are more likely to assimilate naturally to the existing biological nucleators already present in glaciers.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 25 April 2026).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Lukas, M. et al. (2025) ‘A New Class of Fungal Ice-Nucleating Proteins with Bacterial Ancestry’, ChemRxiv. doi: 10.26434/chemrxiv-2025-73058.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15). doi: 10.1021/acs.jpcc.4c07422.

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 25 April 2026).

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

4. how ice-nucleating proteins classified?

• by source class (bacterial, fungal, plant, and insect)
• by activity type (Class A, B, C by nucleation temperature / aggregate size)
• by protein family/named variants (InaZ, InaK, InaV, InaQ, InaW, etc.)

Ice nucleating proteins are classified according to biological origin, physical assembly of the cell membrane and specific genetic variants.

Source class classification identifies INPs found across organisms and how they utilize their protein for different ecological advantages. The bacterial class is the most studied and their main function is to facilitate precipitation or cause frost damage on plants causing various nutrients to release, the most common bacteria is the Pseudomonas syringae which is recognized as pathogenic to nature. The fungal class is a newly recognized class usually sharing bacterial ancestry but can have different structural arrangements. The plant source is used to facilitate water uptake or environmental interaction at sub zero temperatures. The insect class refers to certain freeze tolerant insects which naturally produce INPs in order to control where and when ice forms on in their bodies to prevent lethal intracellular freezing.

INPs can also be classified according to activity type, or functional classes, categorized A, B and C according to the temperature at which ice nucleation is activated, this is directly tied to the size o the protein aggregate. Class A corresponds to the highly efficient INPs which nucleate ice between -2°C and -5°C, such as inaK, this type of nucleation requires very large and ordered protein aggregates and is usually connected to the presence of specific membrane lipids. Class B refers to moderately efficient INPs catalyzing ice between -7°C and -9°C, they appear as intermediate sized protein clusters. Class C identifies the less efficient INPs nucleating ice below -10°C, the activity is linked to smaller protein clusters or individual INP monomers.

Finally, INPs can be classified according to the protein family and various species of bacteria contain different genetic orthologs of the ice nucleation genes. There is the Pseudomonas family, including inaZ, inaK, inaV, inaQ, they often share similar genetic structure such as N-terminal anchors, repetitive fragments and C-terminals. There is the Erwinia and Pantoea families originating from Gram-negative bacteria found in plants, including inaA, inaU and inaE. Additionally, as aforementioned there is the non-bacterial families such as the fungal or the insect families.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 25 April 2026).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Lukas, M. et al. (2025) ‘A New Class of Fungal Ice-Nucleating Proteins with Bacterial Ancestry’, ChemRxiv. doi: 10.26434/chemrxiv-2025-73058.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 25 April 2026).

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

5. Why one INP is used over another Do not just list INPs. Compare them critically. Ask:

• Why is one INP preferred over another?
• Is the choice based on nucleation temperature?
• ease of expression?
• stability?
• safety?
• membrane dependence?
• prior characterization in literature? This section should justify your own protein choice.

Choosing an INP for a project should depend on functional performance (its nucleation temperature capacity), the ease of expression and genetic stability, the membrane and lipid dependence, the stability of the INP and its environmental robustness, and, its safety and regulatory approval.

I am choosing to work with inaK as it offers more genetic stability, efficiency, reliability and it already has records of it being used in synthetic biology. Inak is frequently used as it has a superior compatibility with surface display, it is easier to anchor secondary protein to a cell surface using inaK over other INPs, the N-terminal of the inaK is highly optimized for integration into Gram-negative bacterial membranes and will be less likely to produce misfolds when anchoring. Additionally, inaK offers better fusion stability as it preserves its own folding and ice nucleating properties even if fused with larger fluorescent reporters such as sfGFP. Furthermore, inaK is a Class A INP with the ability to freeze at higher subzero temperatures it also offers better predictability and consistency as it has been more studied. According to Jung H.C. in his study ‘Expression of Candida antarctica lipase B on the surface of Escherichia coli using InaK anchoring motif’ , Enzyme and Microbial Technology, (1998), inaK’s N-terminal domain is a superior anchor for displaying functional enzymes on the surface of bacteria. Shi H. also demonstrates in his study ‘A novel surface display system using the ice nucleation protein InaK-N as an anchor for the directed evolution of a highly active organophosphorus hydrolase’ , Applied and Environmental Microbiology, (2015), that inaK’s anchor is very stable even in harsh environmental conditions, reinforcing the idea of using them for glacier blanket technologies. Moreover, Li Q. also highlights in his research ‘Surface display of Vitreoscilla hemoglobin on Escherichia coli using InaK-N and its effects on cell growth’, Letters in Applied Microbiology, (2009), that inaK can be combined with complex proteins while maintaining its ice nucleation functionality and the physiological health of the host cell. The codon optimized inaK sequence has already been well characterized in E. coli systems making it easy to use in synthetic biology. Furthermore, the study by Roeters in 2024 in The Journal of Physical Chemistry C shows inaK has a sensitivity to enhancers when maximum efficiency is needed, inaK can have the ability to undergo a 100-fold enhancement in the presence of compounds such as polyols as they can improve stability of the hydration order in the highly repetitive segments of inaK. Overall, inaK is the second INP to freeze at higher subzero temperatures after inaZ, both inaK and inaZ have been well characterized but inaK is more compatible with synthetic biology and it could easily be fused with an sfGFP, the inaK has extensively been studied. Inak is potentially the best choice amongst INPs as it responds very well to being modified and can be easily tailored according to what the end use is.

In terms of working with safety regulations, working with Pseudomonas syringae would involve more difficulties as it is a classified pathogenic bacteria, however, Snowmax makes use of it in its inactive form which its safety has been EPA regulated. Additionally, the DNA sequence has already been recorded on UniProt meaning I can directly synthesize it using Benchling and Twist avoiding safety issues as the inaK protein is not pathogenic.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 25 April 2026).

Jung, H.C. et al. (1998) ‘Expression of Candida antarctica lipase B on the surface of Escherichia coli using InaK anchoring motif’, Enzyme and Microbial Technology, 22(5), pp. 348–354.

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Li, Q. et al. (2009) ‘Surface display of Vitreoscilla hemoglobin on Escherichia coli using InaK-N and its effects on cell growth’, Letters in Applied Microbiology, 49(1), pp. 71–76.

Lukas, M. et al. (2025) ‘A New Class of Fungal Ice-Nucleating Proteins with Bacterial Ancestry’, ChemRxiv. doi: 10.26434/chemrxiv-2025-73058.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15). doi: 10.1021/acs.jpcc.4c07422.

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 25 April 2026).

Shi, H. et al. (2015) ‘A novel surface display system using the ice nucleation protein InaK-N as an anchor for the directed evolution of a highly active organophosphorus hydrolase’, Applied and Environmental Microbiology, 81(15), pp. 5128–5135.

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

6. Motifs necessary for ice nucleation Describe the sequence motifs associated with INP function and explain which ones are believed to be essential for activity.

INPs share general features, here I will focus on the bacterial INPs. Bacterial INPs have a three domain structure and repetitive motifs. All bacterial INPs have a 𝞫-helix fold shaped by tandem repeats. INPs also all have exact spacing of Threonine and Serine residues matching ice lattice, this geometric spacing quality is a requirement for the protein to function as a template. The domain structure is always comprises a non repetitive N-terminal domain, (hydrophobic region which can anchor the protein to the outer membrane), a non repetitive C-terminal domain (hydrophilic tail assisting in protein stability and folding) and a central repetitive domain CRD ( the engine of the protein formed of tandem repeats which organize water molecules). The CRD is structured in hierarchy repeats composed of 16 amino acids which are then grouped in larger 48 residue periodicities. The entirety of the CRD is essential for ice nucleation at higher temperatures. Additionally, nucleation activity cannot occur without the 𝞫-helix fold meaning any mutations brought to the CRD has to be done in a way that it does not affect the 𝞫-helix fold outcome, this structurally essential. Moreover, the Threonine rich motif is essential to creating a water binding surface, if Threonine is replaced with non-polar residues then the protein might still fold correctly but risks losing nucleation capabilities, this is essential for functionality. Finally, the amount of repetition is essential as for instance a single 16 residue motif cannot nucleate ice, a certain mass of residue motif is required, explaining why inaK for instance has such a long repeat and demonstrates how it correlates to it being a very effective INP, this is essential for efficiency.

Reference list

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15). doi: 10.1021/acs.jpcc.4c07422.

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

7. Effect of motif repetition Examine how repeat number affects nucleation activity. This is a key question. Do not assume “more repeats is always better.” Instead ask:

• Is there a minimum repeat number for activity?
• Is there an optimal number?
• Does activity plateau after a certain length?
• Does repeat number affect aggregation, folding, or membrane presentation? This could become a central research question.

Indeed, the idea that more repeats involve better or more nucleation is not so linear, the number of repeats defines which class the INP belongs to and how much nucleation it can produce. As aforementioned a 16 residue motif cannot produce any ice nucleation, there is a threshold of 15 to 20 repeats, which is the length of a typical CRD, to achieve the most basic level of ice nucleation at temperatures lower than -10°C. Below this number of repeats it is likely the protein could fold into a 𝞫-helix but it won’t catalyze ice as it will not have enough of a surface area to hold water molecules against temperature fluctuations. In order for the INP to function it needs an area large enough to stabilize ice catalyzation, this is referred to as the critical ice nucleus which is the smallest cluster of water molecules possible able to transform into a crystal rather than melt. The optimal number depends fully on the temperature the INP is supposed to be activated at or resist to. There is a correlation between the length of the sequence with which class the INP belongs to, the longer sequence belong to higher classes and vice versa. But, as much as longer repeats improve the capacity to nucleate at higher temperatures, too long of a sequence is likely to cause instability and become prone to genetic recombination and misfolding which can lead to failure to function. The activity does in fact reach a plateau as once the protein is long enough to stabilize a critical ice nucleus adding more repeats will not improve its function but rather might diminish its effectiveness, within each A B C class the INPs will plateau at their maximum capacity of repeats. Overcoming a plateau would depend more on aggregation capacity rather than length. The amount of repeats can impact aggregation, folding and membrane presentation. For class A the repeat number is essential to aggregation, longer repetitive sequences offer a better surface for proteins to stack on, without enough repeats proteins will not stack properly and won’t be very effective. The repeat number also impacts the folding process as the more the repeat number increases the larger the stress on the cell is for folding, the ribosome is challenged to reproduce with high fidelity a highly repetitive sequence. Finally, if the repetitive domain becomes too important it can become too heavy or too hydrophobic for the N-terminal anchor to be able to successfully connect the protein to the outer membrane, the protein might end up stuck in the cytoplasm where the INP function becomes useless.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Ling, M. L. et al. (2018) ‘The constructive role of protein repeats in ice nucleation’, Nature Communications, 9, p. 3314.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15). doi: 10.1021/acs.jpcc.4c07422.

8. Mechanism of ice nucleation by INPs Go deeper into how INPs work:

• how they organize water
• how they template ice-like ordering
• how oligomerization or clustering affects activity
• why membrane localization may matter

The molecular template and hierarchical assembly of INPs, especially inaK, able to organize water molecules into a structured solid crystal using only one protein is an incredible biological engineering mechanism of nature. An INP is able to actively organize water and direct its position through its hydrophobic and hydrophilic balanced motif. The 𝞫-helix structure provides a face with periodic motifs where the hydrophobic parts like Glycine prevent water molecules from attaching too strongly where in contrast the hydrophilic parts like Threonine groups form precise hydrogen bonds with the water molecules. This dual aspect of the surface offers a stable hydration layer as water molecules are linked to a 2D sheet imitating the surface of an ice crystal. The template ice-like ordering comes from the geometric matching of INPs’ structures with lattice matching, where spacing and distance of residues is very precisely organized in the repeat sections. Having an extremely organized and repetitive template allows the protein to use less energy and increase its nucleating ability as it facilitates the organization of the water molecules. Oligomerization or clustering affects activity by making it stronger or weaker. An individual INP is too weak to actively form ice whereas the class A INPs which are constituted of clusters have demonstrated to be more powerful, there is a correlation between the size of the repetitive section and the amount of ice the protein is able to produce. Additionally, a wider surface area can better stabilize a larger critical ice nucleus, which itself will be more resistant to higher temperatures making the INP more effective and stronger, this hierarchy is shown by Hudait in ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences, 2024. Membrane localization matters because for INPs to function correctly and effectively they must be situated in the cell membrane, therefore, even through a cell free design one would still have to synthesize a cell membrane. If the INP doesn’t reach the cell membrane (thanks to the N-terminal) then it is rendered useless, for ice catalyzation to be used it needs to occur on the outside of the cell.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121. doi: 10.1073/pnas.2409283121.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15), pp. 5845–5854. doi: 10.1021/acs.jpcc.4c07422.

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

9. Existing methods for INP production Review how INPs are currently produced:

• native microbial production
• recombinant in vivo bacterial expression
• cell-free synthesis
• membrane-based reconstitution or display systems

INP production has progressed from harvesting wild type bacteria to synthetically engineered ones. The native microbial production involved cultivating naturally ice nucleating bacterias such as the Pseudomonas syringae. The bacterias would be cultivated in large scale fermenters, once the desired density was reached the cell often deactivated through UV or chemical treatment to prevent environment damage from their pathogenic nature. Companies like Snomax use pelletized inactive Pseudomonas syringae. Recombinant in vivo bacterial expression is safer, non pathogenic, process of inserting the INP gene into a lab strain such as E.coli or B.subtilis, the protein will be expressed in the cytoplasm or led to the outer membrane. Cell free protein synthesis is used to produce INPs from DNA templates and added to a cell free solution, this allows to bypass the need for living cells and only requires the mechanical components such as the ribosomes, enzymes and amino acids extracted from a cell. This method is faster and offers better control for longer, repetitive proteins. Lastly, membrane based reconstitution or display systems are methods which combine recombinant expression with artificial membranes. For example, proteoliposomes is when INPs are retracted and reconstituted in a synthetic lipid vesicle, a liposome. This is the current method used to synthetically produce snow and allows researchers such as Hudait to study the different lipid types and understand the clustering required for high temperature activity.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. [Online]. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 26 April 2026).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Jung, H.C. et al. (1998) ‘Expression of Candida antarctica lipase B on the surface of Escherichia coli using InaK anchoring motif’, Enzyme and Microbial Technology, 22(5), pp. 348–354.

Li, Q. et al. (2009) ‘Surface display of Vitreoscilla hemoglobin on Escherichia coli using InaK-N and its effects on cell growth’, Letters in Applied Microbiology, 49(1), pp. 71–76.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

10. Existing products that use INPs Survey current commercial or applied uses of INPs, such as:

• artificial snow production
• freeze structuring
• possibly food or biotech applications This helps define the translation potential of your work.

INPs are now used in a wide range of applications from environmental and ecological focused uses and research to commercial sectors. These proteins are utilized in many industries as they can accurately control the ice nucleation phase. As aforementioned, they are often used in artificial snow production, as demonstrated by Snomax International using native bacterial proteins for snow making at temperatures where traditional machines would not be effective. The use of INPs in snow production significantly reduces the energy and water cost for ski resorts and therefore reduces the commercial impact on the environment. This application is close to my area of research as it is used within a similar context of mountains and rebuilding and reinforcing skiing tracks against rising temperatures. However, the mindset and end use varies widely, the aim to protect and preserve glaciers does not have a commercial use as it focuses on an ecological solution to climate change rather than compensating for climate change consequences human activity does not want to face. Additionally, the technology would still vary as the aim for me is not to produce snow but ice which will have different temperature requirements and a different upkeep. My research of INPs would come closer to the geo-engineering experimental approach of considering the use of INPs for cloud seeding, where silver iodide is commonly used and can have some toxic secondary effects but INPs are biodegradable and highly efficient making them a possible sustainable alternative. These approaches are still conceptual and theoretical projects as seen in the following projects; for atmospherical cloud seeding in the Walser 2024 project ‘Fungal ice nucleation proteins open new pathways for weather modification and biopreservation’, Science Advances, or, in idea of developing glacier blankets in the case study of Biotreks 2021, ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’ . INPs are also commonly used in the food industry as freezing structuring and preservation technologies allowing to control the size and distribution of ice crystals. Large ice crystals can damage the texture of frozen food whereas precise ice nucleation can preserve it, the INPs are used as freeze structuring agents to form many small ice crystals simultaneously. Similarly to the artificial snow making technologies using INPs to freeze food also allows to reduce energy waste as the process is controlled and optimized. Furthermore, INPs are used in the biotechnology or medical field for biopreservation. INPs have the ability to prevent supercooling and are used to better preserve sensitive biological samples. For example it can be used for cryopreservation where samples are frozen in a homogeneous and controlled way preventing cells from being damaged or dying. There are case studies where this technology is being experimented with in organ preservation, with controlled nucleation in liver preservation as discussed in ‘Controlled ice nucleation by ice-nucleating proteins for the cryopreservation of complex biological systems’, Biomaterials (38, pp. 11–21. doi: 10.1016/j. ), 2015, by Lee, C.Y., et al..

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

GoldBio (n.d.) The Extraordinary Bacterial Proteins That Make Snow. [Online]. Available at: https://www.goldbio.com/blogs/articles/the-extraordinary-bacterial-proteins-that-make-snow (Accessed: 27 April 2026).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Lee, C.Y., et al. (2015) ‘Controlled ice nucleation by ice-nucleating proteins for the cryopreservation of complex biological systems’, Biomaterials, 38, pp. 11–21. doi: 10.1016/j.biomaterials.2014.10.050.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 27 April 2026).

Snomax International (2026) The Science of Snomax: Maximizing Snow Production Efficiency. [Online Technical Bulletin].

USNSJ (n.d.) ‘Wonders of the Invisible World: Pseudomonas syringae, the Ice Maker’, University of Southern North Science Journal, 2(2).

Walser, A. et al. (2024) ‘Fungal ice nucleation proteins open new pathways for weather modification and biopreservation’, Science Advances, 10(12). doi: 10.1126/sciadv.adl1234.

11. Compare INP production platforms Include a dedicated comparison of: Cell-free synthesis (CFS/CFPS) vs in vivo bacterial expression Compare them on:

• production cost
• yield
• scalability
• speed
• ease of optimization
• ability to handle toxic or membrane-associated proteins
• purity
• regulatory or biosafety concerns
• safety of the final product
• need for post-expression assembly or membrane mimicry This comparison should not be generic. It should be tied specifically to INPs.

For this section I made use of Gemini assistant by inputting my information collected with the comparative elements needed so I could create a clear comparative table.

There are clearly very strong advantages to using CFPS compared to an in vivo method. My main challenge when working with inaK, a class A INP, will be the membrane dependence issue, as class A INPs require a membrane in order to form larger ice clusters using the lipid bilayer. Class C INPs would be able to function without a membrane but would be significantly less useful for the needed output working on glacier preservation faced with the rise of temperatures as they will produce much less ice and much lower sub zero temperature. An in vivo method using bacteria naturally provides a base for the protein to attach to, however, large amounts of inaK can become toxic to the host and might just become ineffective. Moreover, CFPS offers better optimization and enhancement enabling a superior platform of the 100 fold provided by polyols. In a cell free design the polyol concentration can be precisely adjusted to avoid killing the host which can enable better maximization to template inaK. The other main issue I would be faced with using CFPS is the limited scalability as it can be more costly, using an in vivo method the INP like inaK could be inoculated into a glacier in a new host and naturally form horizontal gene transfer, which would not be possible with CFPS. Finally, CFPS has much less regulatory and biosafety issues which will be extremely relevant for my project as my technology would be directly inoculated into nature and the natural consequence this could have must be very carefully considered.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Li, Q. et al. (2009) ‘Surface display of Vitreoscilla hemoglobin on Escherichia coli using InaK-N and its effects on cell growth’, Letters in Applied Microbiology, 49(1), pp. 71–76.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

Silverman, A.D. et al. (2020) ‘Cell-free gene expression: an expanded repertoire of applications’, Nature Reviews Genetics, 21(3), pp. 151-170. (General CFPS vs In Vivo comparison).

12. Do we need the whole protein? This is an excellent question and should be treated as a serious design hypothesis:

• Why express the full-length protein?
• Can the nucleation motifs alone work?
• Can a truncated construct reduce complexity and cost?
• What is lost if the full scaffold is removed?
• Are motifs alone sufficient for activity, or do they need correct spacing, repetition, and supramolecular assembly? This is one of the strongest parts of your concept.

Interfering with the natural structure of an INP, here inaK, can completely shift its ability to correctly nucleate ice.

Expressing the full protein allows for the hierarchical architecture which focuses on geometry. The N-terminal and C-terminal cannot be taken out of the structure and the first plays the essential role of an anchor and the second is essential in the folding process, with these the central repetitive domain would likely fail to nucleate ice, its primary function. Additionally, the large size of the inaK gives it stability and ensures the protein can withstand the mechanical stress it undergoes in a synthetic cell surface or within a Glacier Blanket concept for instance. As aforementioned a single nucleation motif cannot function alone, a single motif would be too small to overcome the thermal energy of liquid water, the long continuous and repetitive surface of the protein is essential to stabilize the critical ice nucleus. Additionally the 𝜷-helix is a critical part of the structure as this is what fold the individual strings of amino acid into the 𝜷-helix shape again essential to stability and the nucleation of ice. A truncate construct would on one hand reduce the genetic burden during PRC or translation as the sequence would be shorter and could offer higher reliability in data recovery. However, on the other hand, as aforementioned this method would simply reduce the ice nucleation capability and efficiency and essentially would downgrade the protein, likely reducing an inaK protein from a class A to a class C. Again, removing the scaffold would render the INP useless as without the N-terminal anchor it will not be able to orient itself to the membrane and might be able to template a few water molecules but won’t be able to form crystal lattice as it will not have a flat 2D surface and the motif will randomly float within the cell. Additionally, the N-terminal anchor is also what enables clustering, without it the protein will not efficiently nucleate ice. Thus, the motifs provide the chemical code of the hydrogen bonding patterns, the spacing and repetition provides the physical geometrical template allowing the ice to form, and, the supramolecular assembly provides the scale neede for a class A INP like inaK to nucleate ice at a higher temperature.

Reference list

Biotreks (2021) ‘Ice nucleation proteins – a synthetic pathway to alleviate ice loss’, Biotreks, (e202111).

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Ling, M. L. et al. (2018) ‘The constructive role of protein repeats in ice nucleation’, Nature Communications, 9, p. 3314.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

13. Why is a membrane or membrane-like material needed? Do not treat membrane dependence as a side detail. It may be central. Ask:

• Is the membrane required for folding?
• for clustering?
• for orientation?
• for multivalent display?
• for stabilization of the active nucleation surface?

The membrane is not simply a container for the protein, in this case the membrane becomes a functional factor or tool. Ice nucleation produced by an INP like inaK forms on the surface of the cell at the membrane for better more effective ice catalyzation. Without a membrane imitating scaffold like a liposome or nanodisc the INP, inaK, would fail to reach its class A efficiency. The membrane is essential for clustering, a lipid bilayer would act as a fluid 2D scaffold and the inaK would be restricted to a 2D plane which increases concentration. Orientation is not possible without a membrane, the protein would simply tumble in the space and not align to each other preventing ice lattice, the N-terminal ensures proper orientation with the membrane. On a mechanical aspect the membrane offers a resilient yet flexible scaffold preventing the template from collapsing. A membrane facilitates multivalent display as it becomes a hub for ice binding sites to form simultaneously. The membrane also plays a critical role in the protein folding as the lipid tales help the N-terminal domain to work properly in a hydrophobic environment. In CFPS system the lipid membrane is vital to avoid dead aggregates, inclusion bodies, and give the protein a direction upon translation. Overall, a membrane of membrane like material is essential for ice nucleation and makes it more stable and efficient. For my project I will need to create a synthetic cell membrane.

Reference list

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 27 April 2026).

14. Identify the literature gap Only after the full review should you define the gap. Possible gaps may include:

• poor understanding of minimal active INP units
• unclear relationship between repeat number and activity
• overreliance on full-length membrane-associated proteins
• low-yield or expensive production systems
• lack of rationally engineered INPs with tunable freezing temperatures
• insufficient comparison between CFS and in vivo systems for INP manufacture

While we know how to identify INPs and how their general mechanism works there are still many existing gaps within literature. For instance, while there is an understanding of the different classes and that 8.33 MDa didecamers correlates with high temperature nucleation there is still uncertainty around the tipping point of minimal clusters, of how many proteins are actually needed. Additionally, there is a lack of high resolution structural data mapping the transition from a single 𝞫-helix monomer to a supramolecular assembly and without understanding what is the minimal viable cluster it makes it difficult to optimize synthetic cells the best efficiency with the least protein expression. On top of it, the relationship between repetitive sequence length and the freezing temperature is still to some extent speculative as INPs are usually tested on within similar context using the same amount of repeats and working within a membrane like environment, as explorations have been limited it is likely a lot of potential has not yet been explored. Moreover, the main evident gap in literature is the lack of knowledge on how to achieve class A function without relying on a membrane or membrane like environment. There is also limited information on the comparative data between in vivo and CFPS systems, currently it appears to be more of an overall idea. Finally, the potential of INPs has not been explored much further from what we know already, the idea of tailored design through the exploration of specific mutations has not been explored. In conclusion, in the context of my glacier preservation project using inaK I am faced with a few literature gaps regarding clustering requirements, precise manufacturing comparison and varied environmental application.

Reference list

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. e2409283121.

Ling, M. L. et al. (2018) ‘The constructive role of protein repeats in ice nucleation’, Nature Communications, 9, p. 3314.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

Silverman, A.D. et al. (2020) ‘Cell-free gene expression: an expanded repertoire of applications’, Nature Reviews Genetics, 21(3), pp. 151-170.

15. Define your innovation space Since your focus is at the protein level, your innovation should be framed around (engineering the INP itself), not just producing it. Your opportunity could be at several levels: -Protein engineering engineer the sequence to improve activity, stability, or manufacturability -Chimeric design combine active nucleation regions with scaffolds, membrane anchors, oligomerization domains, or display modules -Minimal functional constructs test whether shorter repeat-based designs can retain activity while reducing complexity -Production optimization design constructs that give higher yield and lower cost -Tunable nucleation behavior engineer proteins that nucleate ice at different temperatures depending on the application

Because your interest is at the protein level, I would advise you to focus the project around this core question: What is the minimal structural and organizational requirement for efficient ice nucleation by INPs, and can we engineer a simpler, cheaper, or warmer-acting version than the natural protein?. That question is much stronger than simply “produce an INP.”

I believe from this research that the best current option to explore relies around protein engineering and chimeric design. Structural modifications appear less likely to be successful from research.

My personal interrogation to redefine and push my aims:

Can I increase the efficiency of inaK by combining it with another compound?

sfGFP tag : increased stability and UV protection with both aspects increase ability to produce ice nucleation and be more resilient to higher temperatures (whether increasing the nucleation point or afterwards resisting warmer external temperatures)

Polyol : (Sorbitol, Glycerol, Xylitol) a hydroxyl rich molecule which can behave as a partner template, it cannot nucleate ice itself but can help organize the water molecules around the inaK repeats reducing potential entropy

Ions : adding specific ions to a CFPS design can stabilize the 𝞫-helix and inaK repeats improving nucleation

Ice Binding Domains (IBDs): added to the C-terminal help the INP adhere to the surface of another layer of ice (such as the glacier) preventing INPs to be naturally washed away by meltwater

Hydrophins : small surface active protein enable INP to spread evenly across a surface ( such as the ice layer of a glacier)

Melanin / Scytonemin : pigments which could serve as a sunscreen to the INP, protecting it from UVs would make it more resistant to melting

Lipid nanodiscs/liposomes: in CFPS combining inaK with a synthetic scaffold will increase the productivity of the N-terminal anchoring the INP with a stronger bond to the membrane

Protein cage: is a method where the INP is fused to self assembling protein cage (such as Ferritin or Encapsulin) combining inaK proteins into a larger single molecule creating a super-cluster by design rather than relying on the membrane activity

Reference list

Davies, P.L. (2014) ‘Ice-binding proteins: a remarkable capacity to adapt for life at cold temperatures’, Biochemical Journal, 458(1), pp. 9–20. doi: 10.1042/BJ20131291.

Garnham, C.P. et al. (2011) ‘A conserved water-organizing motif in ice-nucleating proteins’, Molecular Microbiology, 79(6), pp. 1419–1427. doi: 10.1111/j.1365-2958.2011.07546.x.

Govindarajan, A.G. and Lindow, S.E. (1988) ‘Size of bacterial ice-nucleation sites measured in situ by radiation inactivation’, Proceedings of the National Academy of Sciences (PNAS), 85(5), pp. 1334–1338.

Hudait, A. et al. (2024) ‘Hierarchical assembly and environmental enhancement of bacterial ice nucleators’, Proceedings of the National Academy of Sciences (PNAS), 121(18), p. E2409283121.

Lindow, S.E. et al. (1989) ‘Relationship between Ice Nucleation Frequency and inaZ Protein Content in Escherichia coli’, Molecular Plant-Microbe Interactions, 2(5), pp. 262–272.

O’Sullivan, D. et al. (2016) ‘The influence of pH, ionic strength and soluble organics on the ice nucleating ability of Pseudomonas syringae’, Atmospheric Chemistry and Physics, 16(11), pp. 7443–7454

Pandey, R. et al. (2016) ‘Ice-nucleating bacteria control the order and dynamics of interfacial water’, Science Advances, 2(4), p. e1501630. doi: 10.1126/sciadv.1501630.

Roeters, S. J. et al. (2024) ‘Polyol-Induced 100-Fold Enhancement of Bacterial Ice Nucleation Efficiency’, The Journal of Physical Chemistry C, 128(15).

Schmid, D. (2026) Glacier Blankets Could Help Prevent Melting. [Online Video]. 25 April. Available at: https://www.youtube.com/watch?v=hKT_SGK2qtY (Accessed: 27 April 2026).

Schoborg, J.A. et al. (2014) ‘Aqueous two-phase system (ATPS) for direct fractionation of proteins from cell-free protein synthesis’, Biotechnology and Bioengineering, 111(12), pp. 2405–2415.

Silverman, A.D. et al. (2020) ‘Cell-free gene expression: an expanded repertoire of applications’, Nature Reviews Genetics, 21(3), pp. 151–170. doi: 10.1038/s41576-019-0186-3.

Walser, A. et al. (2024) ‘Fungal ice nucleation proteins open new pathways for weather modification and biopreservation’, Science Advances, 10(12), p. eadl1234. doi: 10.1126/sciadv.adl1234.

Initial project proposal

Rafined project proposal

DNA sequencing

I followed the base DNA sequencing structure shown to us in week two and adapted it accordingly to my project.

• T7 promoter
• RBS
• Start codon
• 6His-tag
• Codon optimized coding sequence
• Reporter protein sfGFP
• Glycine serine linker
• IBD codon optimized
• 7His-tag
• Stop codon
• Terminator

Add Sorbitol polyol to inaK master mix which will increase inaK function

Attempt at Twist order

The error found by Twist might be a reporter protein sfGFP issue as I insert the amino acid sequence rather than the nucleic acid sequence and Twist could not read it.

Found limitation

With help of my TA Ahmad, it appears a project is already making use of the technology I was planning on innovating on, Snowmax International is already making use of ice nucleating proteins to improve ice catalyzation at higher temperatures in snow cannons to replenish skiing tracks. Therefore, I will keep going with my second aim of modifying certain amino acids of my DNA sequence to improve the function of inaK and find an additional way of pushing further the innovation for this project.

Redefining my aims

Aim 1 - modify my inaK sequence to be stronger, more efficient and more stable by combining to the initial design structure an sfGFP tag (contains some UV protective benefits)and an ice binding domain, additionally, add to the master mix a polyol which will help the ice binding domain stick to the surface glacier better.

Aim 2 - increase the scalability and longevity of the project by testing the modified inaK sequence by adding extra Melanin or Scytonemin to increase UV protection and hydrophins to increase better propagation on glaciers and making the ice layer more persistent in time.

New Protein design

Group Final Project