Design

Abstract

The Cellulose Aerogel

Introduction

First discovered in 1931, aerogels are solid foam composed of a network of interconnected nanostructures that exhibit a porosity (non-solid volume) of no less than 50%. Unlike regular gels, aerogels are dry materials with a gas or vacuum in its pores instead of the liquid1.

Aerogels are mostly produced with sol-gel chemistry followed by freeze-drying or critical point drying which creates a 3D porous nanostructure with great physical properties, such as high porosity (~80-99.8%)2. This nanoporosity makes aerogels excellent materials for a broad range of applications, such as biomedical and environmental. Our project explores the new, sustainable way of using aerogel in the construction sector as thermal insulators3.

HESTIA decided to focus on the construction materials to not only open the world of construction to synthetic biology, but also to use the local expertise of EPFL and to address the looming energy crisis. We started by looking at the energy lost in Europe and more specifically Switzerland. More than 70% of the energy used in Swiss homes is for heating alone4. Upgrading our insulation would mean a significant decrease in energetical spending. Due to its properties, aerogels appeared to us as a great basis for our research into the new building materials. Current insulation materials are well-known for their high production’s energy costs5^,6 and the pollution they cause during buildings’ destruction or renovation7. Thus, we wanted to contribute by opening the way for sustainable and easily applicable aerogels as the insulation material of tomorrow.

Cellulose Choice

Why choose cellulose?

Currently, two types of aerogels are studied and can be produced from different precursors: synthetic polymer-based- and bio-based aerogels8. Synthetic polymer-based aerogel are yet more commonly used even though they have poor biodegradability and toxic precursors along with hazardous degradation products. Additionally, some studies revealed that inorganic aerogels possess weak mechanical properties, which restrain their application to the construction scope8. In the last years, focus has been brought to the fabrication of bio-based aerogels from abundant, cost effective, biocompatible as well as biodegradable precursors. Keeping in mind the environmental situation, they also appeared to us as a great opportunity to make innovative sustainable insulative materials with tuned properties.

Different kinds of bio-based aerogels exist such as chitin-, agar-, gelatin- or cellulose aerogels8. Cellulose aerogels show various advantages, in particular low density and high porosity9. In addition, cellulose is the most abundant biopolymer on the planet making it sustainable and easily accessible material9. Thus, we chose to work with cellulose aerogels to innovate in insulation materials.

Plant cellulose vs Bacterial cellulose

Table 1Comparison between Plant Cellulose and Bacterial Cellulose when used for aerogel production

	Plant cellulose	Bacterial cellulose
Advantages	Can easily be bought Could contribute to a circular economy via recycling Aerogels can immediately be produced and tested No need to adjust bacterial cellulose production to environmental and laboratory conditions	Higher purity than plant cellulose therefore produces more performant aerogels No need to harvest it from the environment, it can be produced independently at low cost Higher tendency to polymerise
Disadvantages	Due to impurities, aerogels produced with plant cellulose tend to be less performant than bacterial cellulose aerogels Certain impurities can severely affect the porosity and properties of the aerogel, making the recycling process energy consuming	Starting the production (adjusting the bacterial culture and production) is costly at first Doesn’t allow for a lot of recycling Before optimisation of the production process, bacterial cellulose is costly time-wise

Taking into account all the characteristics of each cellulose type, we decided to use plant cellulose due to its availability, accessibility and to maximize the time we would get to experiment on cellulose aerogels and their properties. Due to the lack of impurities, bacterial cellulose provides more performant aerogels and very detailed literature on how to produce bacterial cellulose already exists9. However, considering time and feasibility, plant cellulose was a better choice for novel experimentation and trials. Any future iGEM team wishing to further the exploration of cellulose aerogels should feel free to look at the Result and Proof of Concept obtained and work with bacterial cellulose.

Production Protocols

Cellulose aerogel production can be divided in three steps2:

• Creating a dispersing cellulose solution

• Gelation through freezing and then thawing the cellulose solution

• Gelation through freezing and then thawing the cellulose solution

Creating a dispersing cellulose solution

To dissolve cellulose, we derived a protocol from literature10, the detailed protocol can be found on our Protocol page. In accordance with our choice of cellulose, plant cellulose powder was used as our cellulose component. The dispersing solution used to dissolve the cellulose consisted of the NaOH and Thiourea salts, with water acting as the initial solvent.

Gelation

After the cellulose solution was obtained, it was frozen for 24 hours to form a solid structure. The frozen solution would then be thawed, and the hydrogel would be obtained. The freezing-thawing process drives the formation of the solid cellulose skeleton and the polymerisation of the cellulose to create a viable 3D structure.

Drying

The most important challenge to obtain an aerogel is to remove the liquid solvent and to replace it with air or a gas solvent without collapsing the pores due to capillary forces and condensing the solid skeleton. The two most common ways to achieve this is through Critical Point Drying with CO₂ or Lyophilisation, also called Freeze Drying.

Critical Point Drying

Critical Point Drying is the evacuation of the liquid solvent within the hydrogel through solvent exchange with liquid CO₂ and then turning the CO₂ into gas by going around the critical point. For this to work, a solvent exchange from the initial solvent (water) to ethanol has to take place before the drying as CO₂ is soluble in ethanol. Afterwards, liquid CO₂ replaces the ethanol. The temperature and pressure conditions are artificially taken beyond the critical point of CO₂, which turns the CO₂ into a supercritical liquid. In this phase, there is no distinction between a liquid and a gas, and no surface tension is applied to the pore walls. As the CO₂ is then taken to conditions where it will become a gas, it leaves the gel without collapsing the pores, resulting in the formation of an aerogel retaining nearly the initial volume.

Freeze Drying

Freeze Drying, also known as Lyophilisation, is the process where the liquid solvent (water) of a material is first frozen and then sublimated under the right temperature and pressure conditions. As the frozen solvent directly turns into gas, the pores do not collapse and the initial volume is mostly maintained. The one important note is that the initial freezing of the water causes the solvent, and then therefore the pore size, to expand, leading to bigger pores in the resulting aerogel.

The Proteins

Introduction

Cellulose aerogels are performant in terms of thermal insulative properties, but are not resistant against water, nor against fire. We therefore seeked to engineer a modular protein coating that would protect the aerogel from environmental conditions. Since proteins are found everywhere in nature, this type of coating preserves the nontoxic and biodegradable properties of the cellulose aerogel - key features in our design.

Waterproofness was the main property we wanted to provide to the aerogel, to avoid its destruction due to the humidity inside walls essential for the insulation material market (see our Human Practices page). The solution we engineered was designed to cover the aerogel with a hydrophobic silk biofilm. We further engineered a modular protein supplementation strategy, so that one could add additional protein coats with a desired property, be that antifungal, fire retardant, a degradation marker or anything else one could envision. To constitute this additional protective layer, we pictured producing a protein coating that would stick to the silk biofilm already introduced on top of the cellulose aerogel.

The Main Protein Domains of the Project

In order to provide new properties to the cellulose aerogel, we searched for protein domains naturally having these desired properties. We decided to focus on three domains which were the most relevant for the scope of our project, each one having a specific function.

These domains, as well as our designed fusion proteins, have been modeled with AlphaFold, check out our Modeling page if you want to know more!

The Silk Domain: the Waterproof Property

Silk proteins demonstrate interesting mechanical properties such as toughness, strength, lightweight, biodegradability and the possibility to produce different morphologies (fibers, foams, capsules, films)11. In addition to this, silk proteins comprise a high percentage of the amino acids glycine, serine and alanine which have an intermediate hydrophobicity12. As we already decided to leverage the insulative and biodegradable properties of the cellulose aerogel, we investigated a way to protect these materials from water as cellulose aerogel is well-known for its hydrophilicity. We tackled this problem by using synthetic biology, while still honoring the sustainable goals of our approach. Therefore, working with silk proteins appeared to us as the ideal biological and nature-inspired solution for protecting the cellulose aerogel from humidity present in buildings.

After looking into many different kinds of silks, we oscillated between green lacewing and spider silk. Spider silk is more studied, but green lacewing silk seemed more suitable for the goals of this project. Green lacewing insects produce two types of silk: one produced by the larvae (cocoon) and the other by adult females (egg-stalk). The adult produced silk acts as a protective shelter and structural support for egg stalks, which are two ideal properties for a waterproof coating for our aerogel12. Thus, we chose to work with green lacewing silk. This became an additional opportunity to bring further innovation as this type of silk is understudied.

In the species Mallada signata, two serine- and glycine-rich proteins (Ma1XB1 and Ma1XB2) have been identified11, both with highly repetitive core domains and small terminal domains. The core domain’s structure is rich in β-sheets with an approximative sheet-length of four amino acids between turns. These form repeating structural units constituting β-helices which have a significant positive correlation with the proteins’ surface hydrophobicity13. A consensus motif for the core domain of Ma1XB2 (named [AS]) had already been generated. Furthermore, a recombinant protein constituted by 8 repetitions of this [AS] module, N[AS]8C, had also already been expressed in E. coli11. As this artificial egg-stalk protein carried similar properties to full length Ma1XB2, we chose to work with it.

N[AS]8C’s characteristics were further tested in various morphologies such as films, capsules, hydrogels and foams12. When N[AS]8C was forming a film, its hydrophobic properties were enhanced. When N[AS]8C formed a film, its hydrophobic properties were enhanced. Therefore, we decided to engineer a unique hydrophobic biofilm able to bind cellulose to create a protective waterproof coating for our final material.

Origin	Mallada signata (Chrysopidae) green lacewing (insect) Recombinant protein11
Type of expression	Recombinantly expressed in E. coli BL21(DE3), insoluble protein Cytoplasmic expression No post-translational modifications
Structure	Length: 592 aa Molecular weight: 53 kDa Secondary structure: small β-sheets with an approximative sheet-length of 4 aa followed by turns Tertiary structure: succession of β-helices formed with 2-3 small β-sheets.
Activity/function of the protein	Strength Extensibility Biocompatibility Biodegradability Surface hydrophobicity
Usefulness in our project	Can form a biofilm acting as a waterproof coating for our cellulose aerogel

The Cellulose Binding Domain (CBD): the Link between the Cellulose Aerogel and the Protein Coating

Since we chose to produce a cellulose aerogel and to enhance its properties by adding a protein coating, we needed to ensure a safe and efficient interaction between the cellulose aerogel and the proteins. Thus, we looked into cellulose binding domains in the iGEM Parts Registry that we could fuse to our proteins.

The Imperial 2014 iGEM team documented a particular cellulose-binding domain (CBDCipA). The PuiChing Macau 2020 iGEM team used their records to fuse the CBDCipA to the SR protein, another protein that we also used in our project. In addition, the Linkoping Sweden 2019 iGEM team pursued the characterisation of CBDCipA. As this particular cellulose binding domain originates from a thermophilic bacteria (which further increases the domain's applications) and was well documented on the iGEM Parts Registry, we decided to build upon the work of those three iGEM teams. Initially we wanted to reuse the useful composite part CBD-SR from PuiChing Macau 2020 as it was, thus we decided to use its CBD sequence.

Origin	Clostridium thermocellum (C. thermocellum) iGEM Registry: Parts BBa_K1321014, BBa_K3503004 and BBa_K3182001 from respectively the Imperial 2014, the Puiching Macau 2020 and the Linkoping Sweden 2019 iGEM teams
Type of expression	Expressed in nature in Clostridium thermocellum Recombinantly expressed in E. coli BL21 (DE3) Cytoplasmic expression
Structure	Length: 238 aa Molecular weight: 25.3 kDa Primary structure: contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of the faces mainly contains planar strips of aromatic and polar residues which may be the carbohydrate binding part. Secondary structure: composed of a nine-stranded beta sandwich with a jelly roll topology
Activity/function of the protein	Strong affinity to cellulose Binds a calcium ion Binds polysaccharides
Usefulness in our project	Make a link between the protein coating and the insulative cellulose aerogel

The SR Domain: Modular Properties

As we designed a potential way to preserve the insulative cellulose aerogel from water, we next sought other augmentations we could bring to the material. Thermal insulation materials can get exposed to many hazards such as fire, mold from fungus or insects. In light of this we realized that while the material was sustainable, it lacked many defenses against normal building hazards. To overcome this issue, we sought to develop a modular protein coating allowing us to provide a unique adaptability and protection to the cellulose aerogel from its surroundings. Through this customisable coating, the material could be made more fire, fungus or insect resistant depending on the specific needs for the building project.

To test our design, we chose to build upon the Mingdao 2015 iGEM team and used SRSF1 protein (SR). The idea came from the composite part from the PuiChing Macau 2020 iGEM team who fused CBDCipA to the SR protein. We discovered this part when looking into the CBD domain and found it relevant for our project. Due to the high content of nitrogen, coming from arginine residues, and phosphorus, stemming from serine phosphorylation by the kinase SRPK1, the SR protein displays fire retardancy properties.

Given the time we had, our primary goal was to prove the modularity and the linking of proteins to the silk biofilm could work. For the fire retardancy associated with SR proteins check out Mingdao 2015 iGEM team page.

Origin	Found on chromosome 17 in Homo Sapiens iGEM Registry: Part BBa_K1608000 from the Mingdao 2015 iGEM team
Type of expression	In nature: expressed in humans Recombinantly: in E. coli BL21 (by Mingdao 2015) Predicted location: intracellular14, cytoplasmic expression mostly
Structure	Length: 248 aa Molecular weight: 27 kDa Secondary structure: 6 antiparallel β-sheets, 2 alpha helices and some random coils14
Activity/function of the protein	Plays a role in preventing exon skipping, ensuring the accuracy of splicing and regulating alternative splicing Interacts with other spliceosomal components via the RS domains Binds to purine-rich RNA sequences May function as an export adapter involved in mRNA nuclear export through the TAP/NXF1 pathway
Usefulness in our project	Test the linking of modular proteins to the silk biofilm for the modularity of the protein coating Fire retardancy potential of the SR protein when phosphorylated could strengthen the aerogel

The Story of our Design Process

Once the protein domains were chosen (Silk, CBD and SR), we designed how they would be linked together to form protein coating of the cellulose aerogel with the desired properties.

A Gigantic Fusion Protein and the Insoluble Silk Protein

We first thought about engineering a single fusion protein CBD-SR-N[AS]8C that would contain the three domains needed, N[AS]8C being the recombinant silk protein introduced before. We had some important questions about this design: is this fusion protein too big? Would this large fusion protein fold correctly? Would it still be hydrophobic? How would we obtain three ordered protein layers as imagined? What could be our alternatives in case of failure?

Fortunately, from the beginning of our design process, we were in contact with Florence Pojer, the head of the Protein Production and Structure Core Facility (PTPSP) of EPFL, and Kelvin Lau, a scientist of this protein facility. They provided us valuable feedback and advice for the design of the project (see our Attributions page). They warned us that such a fusion protein would probably be too big to be correctly expressed and folded, they also noticed that the silk protein was insoluble in E. coli and its purification would mean unfolding of the whole complex. The insolubility issue was confirmed in the silk purification protocol described in Felix Bauer’s thesis 12 in which acidic conditions were used, indicating that the silk protein gets unfolded and has to be refolded after this step in order to be functional. This would lead to ~90% loss of the desired proteins upon refolding, according to the PTPSP experts, a problem for our large scale material coating goal.

We therefore needed to solve the insolubility problem of our silk protein to avoid protein denaturation upon purification. A solubility tag was the first thing we thought of. We had the option of using common solubility tags such as GST, but we also discovered a NT* solubility tag inspired from the N-terminal domain of spider silks that had been successfully fused to insoluble proteins, bringing higher yields of soluble fusion proteins compared to other conventional tags15. Since we also wanted to express a silk fusion protein, we thought this NT* tag use would be very relevant.

Another idea to additionally improve the solubility of our protein was to reduce the number of repetitive modules of recombinant green lacewing silk: instead of 8 modules, test if 4 modules would be enough to preserve the hydrophobic property of silk and form a silk biofilm. This idea came from the fact that recombinant spider silks composed of 4 repetitive modules have already successfully been expressed in E. coli and showed similar properties to native spider silk16, suggesting that 4 modules derived from recombinant green lacewing silk could be enough to provide the properties we sought.

A Silk Binding Domain?

After having found possible solutions to tackle the insolubility of the silk issue, we had to find a way to link the silk protein to the CBD-SR part, since the big fusion protein idea was excluded. The first thing we thought of, in the same spirit as the cellulose binding domain, was to find a silk binding domain to add to the C-terminus of the CBD-SR part. By searching both in the previous iGEM parts and in the scientific literature, we found a paper which presented a method to select silk binding peptides from silkworm-derived silk fibroin fibers17. However, by continuing the search for a silk binding domain, we found a paper describing a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains18. It presented the biotin-binding domain M4, a streptavidin monomer already characterized in other studies19, which allowed the production of soluble silk fusion proteins and chemically stable silk films. Adding the M4 domain to the N-terminus of our silk domain would therefore be sufficient to biotinylate the SR protein to allow the binding between them.

Due to time restriction, we knew from the beginning that it was not possible to test all these options. Because the generation and the screening of peptide ligands could lead to the selection of non optimal peptides, and because the biotin-streptavidin is one of the strongest known non-covalent interactions20, we chose this linking method for our project. Indeed, this interaction being relatively standard in biology, it would allow an easier protein change for the modular coating. For the biotinylation of the SR protein, we decided to add to its C-terminus an Avitag, that can be biotinylated at a specific lysine either in vivo or in vitro21. Overall this solution suggests two recombinant proteins that would be linked by a biotin-streptavidin interaction. The SR protein would have an Avitag that could be biotinylated, and the silk fusion protein would contain the biotin binding domain, a streptavidin monomer.

Additionally, we thought of adding one of the [AS] modules to the C-terminus of the CBD-SR fusion protein with a possibility of its intercalation into the biofilm formed by silk fusion protein of 4 or 8 [AS] repeats. This assumption was based on the fact that repetitive silk modules are able to polymerize upon biofilm formation. Thus, we decided to test this hypothesis as a second approach to the project.

The Order of Layers

Once we decided on the best suited linking approaches between the two fusion proteins, new considerations came to our mind. First of all, we realized that the silk biofilm did not have to necessarily go on top of the coating, it could also be in between the Cellulose Binding Domain (CBD) and the SR domain. In that case we would have to separate the composite iGEM part CBD-SR (from PuiChing Macau 2020 team) into two and replace the SR sequence fused to CBD by the silk sequence. This new idea of inverting the silk and SR domains quickly appeared as the best solution. Indeed, when thinking about functionality of our material, it made much more sense to put the silk biofilm close to the cellulose aerogel since the biofilm is meant to protect the aerogel from water. Moreover, the fire retardant property is more useful if SR is displayed at the surface of the material so that if there is a fire, the SR will immediately slow down the fire on the material, instead of having the whole silk biofilm burnt before the SR actually becomes useful.

In addition to this, we also wanted to provide more modularity to our design, in order to enable future improvements upon our project. This is why putting the SR domain on top layer (as the modulable part of our project), made more sense. Indeed, the core part of our protein coating being the hydrophobic silk biofilm, we could even propose a product with only a cellulose aerogel covered with silk as a base. Then, it would be possible to add a specific property to the aerogel by coating the silk biofilm with the corresponding protein. We did not confirm fire retardant property of the SR proteins relying on prior studies22,23. Due to all the factors we have described, we decided to alter our previous design: there would be first the CBD directly bound to the aerogel, then the silk biofilm fused to CBD and finally a modular protein (SR in our case) on top of the coating linked to the silk biofilm via biotin-streptavidin interactions (or via cross linking of repetitive silk protein modules).

Our Final Approaches

After the design process described above, we came up with two different approaches combining the aerogel with the proteins which we considered to be most realistically achievable and closest to the intended functionality of our insulative material. Figure 7 illustrates these two approaches.

Approach 1: Biotin-streptavidin Linkage of Silk and Modular Protein

Our preferred approach was linking silk protein to the SR protein by using the strong biotin-streptavidin interaction (fig. 7). Therefore we prioritized this for the wet lab experiments and modeling.

This approach included two main plasmid constructs: 01a and 01b, corresponding to the silk and SR fusion proteins of the coating. The third construct, 03a, was used as a control for the experiments. The 01a construct corresponds to the mSA-silk-CBD protein containing 4 silk [AS] modules and is directly in contact with the cellulose aerogel. The 01b construct corresponds to the SR-Avitag protein and is on top of the protein coating. The presence of Avitag in SR fusion protein was added for providing biotinylation by BirA enzyme. Thereby, the monomeric streptavidin (mSA) present in the silk fusion protein enables the binding between 01a and 01b fusion proteins through the interaction with the biotinylated Avitag.

We designed a control construct for 01a, called 03a, replacing the silk by the green fluorescent protein (GFP). Presence of GFP was used in preliminary assessment of the protein purification experiments, but also downstream, for assessing the interaction of CBD with cellulose aerogel by measuring fluorescence.

Table 2Recapitulative table of the nomenclature for the Approach 1 that was successfully built and tested.For the sake of simplicity, we voluntarily made simplifications when mentioning our proteins. For example, “silk” implicitly refers to “N[AS]4C” and the 10xHis-tag is omitted in the names.

Plasmid name	Protein full name	Protein simplified name
01a	mSA-silk-CBD	Silk fusion protein
01b	SR-Avitag	SR fusion protein
03a	mSA-GFP-CBD	GFP fusion protein

Approach 2: Linkage of Silk and Modular Protein with Silk Repetitive Modules

The second approach for crosslinking silk fusion protein and modular protein was based on the ability of silk’s repeat units to form biofilm when mixed together12. Lead by that, we fused one repeat domain of silk protein to the SR protein, expecting biofilm formation when SR fusion protein is mixed with silk fusion protein upon aerogel coating.

This approach included two constructs: 02a and 02b corresponding to the two fusion proteins of the coating. The 02a construct corresponds to the silk-CBD protein, it is the equivalent to 01a but contains 8 silk [AS] modules instead and no streptavidin monomer (mSA). Since mSA was used as a solubility tag in addition to the biotin-streptavidin linking function, a GST tag was added to 02a plasmid, it would be proteolytically cleaved after purification and thus would not be present in the protein coating. The 02b construct corresponds to the SR-AS protein and is the equivalent to 01b, but instead the Avitag for biotinylation was replaced by a silk AS module.

Engineering Choices

Once we decided on the approaches we wanted to go for, we needed to choose the expression system: the model organism (chassis) and the expression vector. We also had to design the DNA parts to insert in the vector, corresponding to the fusion proteins of our final approaches.

The Expression System

We opted for E. coli as a chassis to produce the recombinant proteins, due to its fast growth, ease of genetic manipulation and potential for industrial scale up24. Since the recombinant proteins do not require post translational modifications, such as glycosylation, nor need to be secreted, a bacterial chassis was sufficient for our purpose. Besides, previously reported production of the recombinant green lacewing silk has been optimized in E. coli11 and the iGEM teams Mingdao 2015 and PuiChing Macau 2020 also used E. coli for successful expression of SR and CBD-SR proteins.

We decided to work with the inducible pET expression system in BL21(DE3) E. coli strain. BL21(DE3) is an E. coli strain commonly used with pET expression system, also called the T7 RNA polymerase system. The gene of interest is under the control of the T7 promoter which is only recognized by the T7 RNA polymerase. In turn, T7 expression is under the control of a lac promoter, therefore inducible by IPTG25. We opted for the pET28a vector as a backbone for inducible expression of our gene constructs, because the pET28 vector was previously used to produce the recombinant silk protein N[AS]8C11.

The different Gene Constructs: a Modular Design

The plasmid backbone and genes insertion

For the plasmids design, we used the backbone of the pET28a-sfGFP plasmid from Addgene and replaced the DNA sequence of sfGFP by the DNA sequences of our domains of interest. We designed five constructs corresponding to Approach 1 and Approach 2 (fig. 8). To obtain highly flexible, modular design, we designed linkers with restriction sites between the fused domains. When designing our iGEM parts, we made sure that they were compatible with the BioBrick assembly standard of iGEM and we removed the illegal restriction sites. Furthermore, additional changes were made to the sequences, such as the addition of the purification tag. For the in-detail information about these constructs, check out the end of this section (fig. 9 and 10). If you want to read more about all the basic and composite parts we created, check out our Parts page!

The His-tags: protein purification and identification with Western blot

For the purification of our fusion proteins, we chose His-tag purification, a widely employed purification method that isolates proteins fused to a polyhistidine (generally six to ten) tag. One advantage of using this purification technique was that His-tags are small and usually do not impede the activity of the fusion protein26. Also, we opted for the tag which doesn’t require additional steps, such as proteolytic cleavage, except for 02b (SR-AS-TEV-10xHis), because the AS module was supposed to interact with the silk domain in 02a (N[AS]8C-CBD-10xHis) and the presence of the His-tag could interfere with the biofilm formation ability.

Having the same tag for all the constructs enabled us to use the same detection method - Western blot for detecting His-tag with monoclonal anti-His antibody. The same detection method was done by the PuiChing Macau 2020 iGEM team, further confirming our choice of detection.

The solubility tags: tackling the issue of the insolubility of the silk

One of our major concerns was the potential insolubility of the recombinant silk. Therefore, we included in the design of the silk fusion protein a solubility tag. The solubility tag was attached to the N-terminus of the fusion protein for allowing proper co-translational 3D folding.

For Approach 1 of our design, we discovered the monomeric streptavidin mSA (which we added to provide linkage via biotin-streptavidin interaction) acts as a solubility tag when fused to spider silk18. However, for Approach 2 we decided to use an N-terminal solubility tag, uncertain that the presence of the CBD domain was sufficient to solubilize the entire protein. Two solubility tags seemed appropriate for our purpose - NT* tag and GST tag. We focused on the GST tag, since it could also serve as a purification tag, if necessary. Since GST tag is quite large, we added an HRV 3C cleavage site (LEVLFQ|GP) between GST and the silk N[AS]8C to cleave off the GST tag after the 02a purification.

The linkers choice: adding modularity to the design

As previously stated, to provide a modular structure, we designed specific linkers in fusion proteins design, named linker A and linker B. Linkers were designed to be of the length to allow optimal flexibility between the domains. Also, linkers included specific restriction sites: a key feature of our modular protein design. Linker A contained AscI restriction site, while linker B contained BstBI restriction site. Another important restriction site was PmeI restriction site proximal to the promoter.

The modularity was based on shuffling of the domain sequences among the different constructs, enabling future testing of other combinations and order of protein domains.

For example, one could switch the silk recombinant proteins of construct 01a and 02a by using the restriction enzymes AscI and BstBI present in linkers A and B, enabling to test the optimal number of silk [AS] modules for each linking approach.

Another idea would be to use the restriction enzymes PmeI and BstBI in constructs 01a and 01b, in order to switch the SR domain with the mSA-silk, enabling to test the system where the SR is linked to CBD and the silk biofilm is on top of the protein coating.

Table 3Summary of the restriction sites used for the modular cloning, their location and in which construct they were present.

Restriction site	PmeI	AscI	BstBI	SalI
Location	Between the RBS and the beginning of the insert	Within linker A. Example: In 01a, between mSA and silk	Within linker B. Example: In 01a, between silk and CBD	Before the 10xHis-tag at the end of the insert. Example: In 01a, after CBD
Construct in which the restriction site is present	01a, 01b, 03a, 02a and 02b (all constructs)	01a, 03a and 02a (all silk-like constructs with CBD)	01a, 01b, 03a, 02a and 02b (all constructs)	01a, 01b, 03a, 02a and 02b (all constructs)

The biotinylation method

In Approach 1, the linkage of the two protein layers on top of the aerogel was biotinylation based. Between chemical and enzymatic biotinylation, we opted for the enzymatic one and added an Avitag at the C-terminus of the SR domain before the polyhistidine-tag. Biotinylation could be done in vitro after the expression and purification of the SR-Avitag (01b) protein by adding biotin and BirA enzyme27. Alternatively, biotinylation could be performed in vivo by the endogenously expressed BirA in E. coli21).

Final designed plasmid maps

Outlook

This last section provides ideas to build on our HESTIA project thanks to its modularity and proposes improvements that could be done to strengthen some aspects of the project.

Modularity in our Design

Modularity in the Protein Coating: Replacing the SR Domain

Beyond the sustainable aspect, the modularity through the protective protein coating is another essential innovation we brought into our insulation material. Indeed, we wished to show that the purification of a fusion protein with an Avitag is possible and that the linking between the latter and the recombinant silk biofilm is feasible thanks to biotin-streptavidin interaction. In our case, we worked with an SR protein which shows fire retardancy properties when phosphorylated by a particular kinase (SRPK1). Therefore, a potential improvement of our project in the future could be to phosphorylate the SR protein and prove its fire retardancy properties on top of our cellulose aerogel.

Besides the opportunity to experiment fire retardancy using phosphorylated SR proteins as a protein coating, we could imagine working with other kinds of proteins. These could bring additional properties to our insulative material in order to protect it more from various hazards or to add novel innovative features. Our constructs design allows us to quickly generate and test novel proteins thanks to the restriction sites added between each genetic element. Some of the ideas for interesting additional features of our cellulose aerogel were fungus resistance 28, to protect it from mold inside walls or a color indicator, which could report on degradation of the material suggesting it should be replaced.

If we can envision producing a protein coating with other fusion proteins than SR, we could also imagine combining all of them together! We could think of adding varying ratios for each protein depending on infrastructure exact needs. The association of proteins bringing different additional features to our material would also constitute a more protective coating. It could increase its lifetime by making it more resistant while improving its adaptability to different environments but nonetheless maintaining biodegradability.

Modularity in the Insulative Material: Replacing the Cellulose Binding Domain

The modularity has an essential role in our project and does not only apply to the protein coating. Actually, we could consider keeping the waterproof silk biofilm and the modular protein coating while switching the insulative material itself. Yet, to allow this modification, a replacement of the CBDCipA protein domain would be required. Finding corresponding protein domains to each material is fundamental to manage the interaction between the silk biofilm and the material itself. This adaptation could bring more sustainability and adjustability to nowadays insulation materials.

Moreover, we could also apply the combination of the waterproof silk biofilm and the modular protein coating to other construction materials, or even materials used in completely different applications. It might increase their adaptability to the environmental hazards they could face.

Biodegradability of our Insulative Aerogel

Biodegradability of our insulative material is one aspect of the project we wish we could have investigated further if we had more time. Indeed, we put a lot of effort into engineering a material made out of natural components such as cellulose and silk proteins and that would therefore be biodegradable and not harmful to the environment and humans. However, we did not have sufficient time to determine the lifetime of such a biodegradable material, meant to last decades within walls.

One could follow the suggestions from Michka Mélo, Margot Wendling and Juliane Miane who proposed to roughly estimate the lifetime of the cellulose aerogel by looking at the lifetime of other materials made out of cellulose, such as cereal straw, taking into account the lifetime of our material would be increased due to its stable, aerogel structure.

However, it would be interesting and more precise to model the biodegradation rate of the cellulose aerogel with a protein coating depending on environmental conditions and the proteins used for the coating. Along this line, it would be important to determine if the proposed insulative material indeed decomposes well in nature (after being properly disposed of), but has enough stability to stay functional during its intended lifetime. As an alternative to modeling, one could also produce more of these protein coated aerogels and assess over several months or years whether this material preserves its properties over time.

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