Team:HKUST/Engineering
Design
Background
What is hagfish slime intermediate filament?
Intermediate filament, with its protein nature and orientation, usually plays an important role in cytoskeleton for maintenance in the shape of the cells. However, in hagfish slime intermediate filaments, they are secreted out to the extracellular environment. Hagfish slime intermediate filament (IF) is originated from hagfish slime, together with mucin vesicles, hagfish IF forms a slime mass which entrains a large volume of sea water. Upon predation, hagfish could release large amounts of slime to clog the gills of predators as a defence mechanism [1]. There are several kinds of species of hagfish. Throughout our project, we focus on the IF from Pacific hagfish (Eptatretus stoutii).
Hagfish slime threads are composed of bundles of IF. IF consists of 2 subunits, alpha (ɑ) and gamma (ɣ) keratin filament. Both subunits possess similar molecular weight and size. ɑ-keratin filament and ɣ-keratin filament will undergo self-assembly to form a ɑ-helical coiled-coil protein structure. This form of IF is very soft and highly extensible, but has little stiffness [2].
And upon draw-processing (pulling the fibers with a certain strain), the ɑ-helical coiled-coil structure will break open and combine with neighbouring strands. This transits the coiled-coil structure into a structure called β-sheet. And it is this transition that grants the fibres with ultra-high stiffness which is comparable to spider dragline silk.
Why hagfish slime intermediate filament?
In our search for possible candidates as a textile fiber material, we compared their extensiveness, tensile strength, the ease of production in a bacterial chassis, and other unique characteristics.
Drawn hagfish slime threads’ textile strength is comparable to spider dragline silk, and its high extensibility makes it a strong candidate.
Spider dragline silk and cocoon silk exhibit impressive physical performance in a dry environment. However, they undergo a unique process, supercontraction, in wet state, in which the fibers will shrink and contract in length. The contraction of spider silk could lead to a 1000-fold decrease (~10Gpa to ~10MPa) in stiffness, and this is less desirable for a textile product. Hagfish slime thread experiences a similar contraction process. However it could be mitigated by extending the draw processing time or by chemical cross-linking [2].
For decades, spider silks have been facing a fundamental challenge in recombinant silk production. The large and repetitive spider silk protein genes are unfavourable to be expressed in bacteria or yeast. Last year’s high school grand prize winner GreatBay_SZ successfully expressed recombinant spider silk using 2 repetitive sequences only. This greatly improved the possibility for spider silk production in bacterial chassis. However, there is yet insufficient evidence supporting that the recombinant spider silk with only 2 repetitive sequences could improve or retain its high material performance. We wanted to avoid changing the amino acids sequence while trying to retain the physical properties of the fibers.
Hagfish slime IF, with the ability to assemble autonomously while increment to stiffness is possible by draw-processing and chemical cross-linking, possess small in size and can be easily synthesised and with the stability to withstand unstable sea water conditions, make it our target candidate as a potential material for textile use.
| Textile strength; Young’s Modulus (GPa) |
Extensibility; Breaking strain |
Ease of production in a bacterial chassis | Unique characteristics | |
| Drawn hagfish slime threads | ~1.1 [2] | ~36% [2] | / | Supercontraction in wet state, which leads to a decrease in stiffness. Could be mitigated by increasing drawing time and chemical cross-link |
| Spider dragline silk | ~1.2 | ~26% [3] | Inefficient production due to large and repetitive sequence | Supercontraction in wet state, which leads to a decrease in stiffness |
| Cocoon silk (species Bombyx mori) | ~0.5 [4] | ~20% [5] | / | Supercontraction in wet state, which leads to a decrease in stiffness |
Comparison of the key properties of biological fibers.
What are chromoproteins?
Chromoproteins are a class of proteins that can give out colour. Unlike fluorescent proteins which absorb UV-light, chromoproteins absorb light in the visible region and emit a specific range of visible light only, which could be observed with the naked eye.
Why chromoproteins?
Chromoproteins are encoded by a single gene only, this allows protein expression to be relatively easy compared to engineering the entire complex metabolic pathway of organic dyes. This simpler dyeing method introduces less variables to the experiments, and results in greater reproducibility of experiments, as well as easier analysis of the results. In natural dyes, dyeing instability due to color fading may occur [6]. Chromoproteins are less well characterised in this aspect, therefore, we will try to determine whether chromoproteins face the same problem as natural dye.
Project Goals / Targets / Principles
Inspired by the 2014 UCC team who attempted to produce recombinant hagfish intermediate filament (IF), we realized there is great potential in hagfish filaments. That’s why we propose a mass production platform for recombinant hagfish IF. We want to provide the foundational basis for turning it into a viable textile material.
While researching solutions to the water pollution problems caused by textile microfibres and the dyeing industry, we realized that as iGEM participants and as scientists in training, we must focus on sustainable development and how our actions will impact the society and environment for subsequent generations.
Therefore, our major goals for this project can be summarized as the following 3 elements of sustainability:
| 1. Environmental | 2. Economic | 3. Social |
| Sought to solve environmental problems caused by microfibre & dyeing using biological materials to reduce our reliance on polluting manufacturing processes. | Reduce cost by proposing a mass production system for hagfish filaments. | Reach out to the textile industry to find out about the biggest obstacles. Creating novel future textile materials with amazing mechanical properties & vivid colours |
Our sustainable Textile System Goals , (1.) Environmental: we hope to solve microfibres & toxic dye problems by replacing with biodegradable and non-toxic raw Materials for fibres & Dye, (2.) Economic: Our fiber production system lay foundational work on large scale productions of hagfish slime IF, and thus reducing cost. (3.) Social: By maintaining sustainability in environmental and economic development, consumers can enjoy high quality novel textile materials with incredible mechanical properties and vivid colours.
Project Design Overview
Previous efforts in synthesising recombinant hagfish fibres have yielded fibers of mediocre strength and quantity [7] [8]. We highlight three (3) major areas of improvement:
1. Fibre Yield
2. Colour
3. Fibre Quality
All of which are crucial if we are to produce a viable textile material for the clothings industry.
Our mass production system is comprised of five (5) aspects:
| Aspects in Our System | Our Design |
| 1. Fundamental research on Hagfish IF |
WET LAB Expression of Hagfish IF in vitro DRY LAB 3D modelling of Basic structure of IF subunit Dimer Model |
| 2. Expression |
WET LAB
Expression methods : Inclusion Bodies VS Extracellular secretion Comparing promoters : K880005 VS T7 Promoter |
| 3. Purification |
WET LAB
T3SS Purification |
| 4. Dyeing |
WET LAB
DRY LAB 3D structure prediction of linking Chromoprotein at different terminal of IF subunits |
| 5. Fibre Formation |
DRY LAB
Stress Model Polymer formation model |
1. Fundamental research on Hagfish IF
In our fundamental research, we focused on the feasibility of synthesizing recombinant hagfish IF and developing the basis for structural modelling of IF proteins.
E. coli is our chassis for expressing the gene sequences of the 2 subunits, alpha and gamma of hagfish slime IF. According to previous teams’ (2014 UCC Ireland) results, it is expected that we will be able to produce synthetic hagfish IF visible under an optical microscope.
We predicted the structures of the 2 subunits of hagfish IF via homology modeling and ab initio modelling. Molecular dynamics simulations are then used to predict the hagfish IF dimer’s 3D protein structure.
2. Expression
We employ several different methods to increase the yield of hagfish IF proteins. It is hoped the addition of a signal peptide for extracellular secretion of target proteins will increase the yield of hagfish IF compared to the traditional method of protein extraction, which involves formation of inclusion bodies followed by cell-disruption. In addition, 2 different high expression strong promoter and RBS sets would be tested to compare the expression rates.
3. Purification
T3SS purification system is employed for efficient purification of hagfish IF subunits, subunits are ejected from the cell to the collection chamber. The usage of T3ss purification system is a cheaper approach to his-tag purification.
4. Dyeing
Various methods for dyeing hagfish IF with chromoproteins are explored. We attempt to link chromoproteins at either the N-termini or C-termini of each of our alpha and gamma hagfish IF subunits to pre-dye the hagfish IF threads. Alternative dyeing methods will be researched for a later stage.
We employ the subtractive colour mixing with three primary colours of chromoproteins (red, blue, and yellow) to expand the colour variations of IF fibres.
5. Fibre Formation
Molecular dynamics simulation is used for predicting the dimer formation of two Hagfish IF subunits with different modifications, such as linking chromoproteins with IF at the N-termini.
In addition, stress models which simulate IF fibres structural changes under stress are performed. This can help visualize the alpha to beta transition of IF during draw-processing, a process that can strengthen the mechanical properties of IF.
To further stimulate the structure of hagfish IF in reality, a polymer formation model of IF will be created in phase 2.
Timeline
A summary of actions to be taken during different phases of our project is provided below:
1. In phase one, we focus on fundamental research: synthesizing the intermediate filament, providing modelling basis for dry lab, and brainstorming.
2. In phase two, we focus on exploring different methods for dyeing and purification of hagfish IF, as well as developing hardware and experimental improvements. More experimental and modeling results will be presented.
Unfortunately, we cannot conduct laboratory work due to Covid-19, thus we don't have enough time before the wiki freeze for synthesizing hagfish IF protein subunits. It is hoped that the situation would improve this winter (2020) that we might be able to do experiments to prove our concepts.
Detailed Project Design
1. Fundamental research (Focus on circuit)
Figure (1) This construct is designed for expressing and producing our alpha and gamma subunits using the T7 promoter and terminator set. T7 is used because we want to try and replicate previous experiment results.
For fundamental research, we first focus on synthesizing recombinant hagfish IF in vitro.
Our target is to replicate the previous 2014 team's (UCC Ireland) results in the lab.
Hagfish (Eptatretus stoutii) Thread biopolymer filament, including alpha (BBa_K3581000) and gamma (BBa_K3581001), is already codon optimized for e coli expression by the 2014 UCC team.
We first clone the 2 subunits from 2014 teams into the high copy pSB1-C3 vector separately. Strong promoter (T7 promoter BBa_I712074) and strong RBS combination (T7 RBS BBa_B0030) is used to ensure high expression level, terminated by T7 terminator (BBa_B0016).
The reason for choosing T7 parts for our expression is to replicate results of a previous experimental data (2014 UCC) which also used T7 parts by secreting gamma subunits of hagfish intermediate filaments.
6x His tag which is inserted in the C terminus of protein to allow purification and identification.A TEV protease cleavage site (amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)) is placed upstream of the 6xHis-tag to facilitate tag removal.
Each plasmid is being transformed into separate e. coli culture (Strain : DH5Alpha). For more details about experimental protocols , please visit Wet Lab.
We predicted the structures of the 2 subunits of hagfish IF via ab initio modelling, molecular dynamics simulations are then used to predict the hagfish IF dimer’s 3D protein structure.
For more details about modelling, please visit Dry Lab.
2. Expression
To increase protein yield, 2 strong constitutive promoters with RBS, BBa_K880005 and BBa_I712074, are incorporated in the construct. Comparison of the protein yield of the 2 promoters can help us determine which construct is more suitable to maximise protein yield.
The usual method of collection of hagfish intermediate filament (IF) is as follows: subunits are first expressed in the cytoplasm inside E. coli. The insoluble form of subunits will aggregate and lead to the formation of inclusion bodies inside the cell. Hagfish IF subunits are then retrieved by cell lysis, where the bacterial cell is disrupted and all cellular contents inside are released; upon further purification, pure hagfish IF subunits could be collected.
However, results from previous research using this approach shows that protein expression yield for hagfish IF is low, in the range of 40-50 mg per liter culture [9]. This yield is far from adequate for producing a viable fabric. Inclusion body aggregation might increase the metabolic load, which could decrease protein expression efficiency [10]. Not to mention the whole process is labor intensive.
Therefore we propose an alternative approach to boost the efficiency of hagfish IF expression and collection. We incorporated a signal peptide, either HlyA (BBa_K3581003) or TorA (BBa_K3114005) into our circuit downstream of our IF subunits at the C-terminus. These 2 signal peptides are chosen for comparison because each has its own advantages and limitations.
(1) Signal peptide HlyA (BBa_K3581003) facilitates protein secretion via the type 1 secretion pathway, allowing our hagfish subunits to be transported from cytoplasm to extracellular space directly. However, HlyA is not cleaved by the bacterial cell when it is secreted out. It has to be cleaved out along before purification by Ni-NTA chromatography so that the his-tag would interact properly with the resin.
(2) Another signal peptide TorA (BBa_K3114005) facilitates protein secretion via the TorA secretion system into the periplasm. Osmotic shock can then be applied to release the periplasmic proteins. In contrast with HlyA, TorA will be cleaved by the cell during secretion. The periplasmic proteins can be used for Ni-NTA chromatography directly.
The advantage of incorporating a signal peptide is that e. coli are not killed but are instead retained for continuous production of fiber proteins while also providing a simple collection process. We hope that by comparing different protein expressions and collection methods, a more practical approach could be determined.
The signal peptides are flanked with HindIII cut sites. This allows us to convert the mode of collection from protein secretion back to the conventional approach by digestion followed by ligation. Therefore we could use the same construct for different experiments.
Figure (2). Inclusion body approach requires cell lysis which kills the cell. However, using a signal peptide, only our IFs are secreted out of the cell
Figure (3) IF expression without signal peptide , inclusion body. Here we will use T7 promoter set for both alpha and gamma subunits
Figure (4) IF expression with signal peptide, extracellular secretion. Here we will use T7 promoter set for both alpha and gamma subunits
Comparing promoters : K880005 VS T7 Promoter
Figure (5) IF expression comparing promoters, T7 promoter. Here alpha and gamma subunits are expressed using the T7 promoter set by inclusion body expression method.
Figure (6) IF expression comparing promoters, K880005. Here alpha and gamma subunits are expressed using the K880005 promoter set by inclusion body expression method.
3. Purification
Our hagfish intermediate filaments (IF) would be of no use to the textiles industry if there is no way to efficiently mass produce it. In previous sections we have described protein expression methods that could produce IF proteins continuously. Here we propose a purification method that could save us time and money. Our proposed implementation is largely inspired by the purification system developed by Team: UCopenhagen 2018, which has its theoretical basis in the bacterial type III secretion system (T3SS).
Overview of the T3SS purification system
Type III secretion system (T3SS) are utilized by many gram-negative pathogens (which includes e. coli) to detect eukaryotic organisms and secrete proteins into them. In T3SS, a structure termed injectisome is a double-membrane-spanning syringe-like structure, which roughly divides into three parts: an extracellular needle-like appendage, associated cytoplasmic and membrane-bound secretion machinery. All three parts work collectively to deliver effector proteins into the cytosol of the eukaryotic host cell. Usually, the protein is secreted in an unfolded conformation, and the secretion requires both ATP and proton-motive force. It also involves an N-terminal secretion signal to guide the effector protein through the injectisome.
T3SS-based purification system in our project
To implement the T3SS purification system into our project, two major steps are involved:
1. Insertion of a T3SS signal sequence in our genetic construct:
We will insert a T3SS signal sequence immediately after the ribosome-binding site (RBS) of our circuit, so that the IF protein expressed would be attached to a T3SS signal peptide. The signal peptide would then guide our IF to reach the injectisome.
2. Transforming Synthetic Injector E. coli (SIEC) with IF subunit gene:
The T3SS genes will be incorporated into the bacterial genome, forming the SIEC. And the subunits gene will be carried by the plasmid vectors. The transformed cells will adsorbed on an artificial membrane via the action of the injectisome. The injectisome would then penetrate the artificial membrane. This allows our IF subunits, tagged with the T3SS signal peptide, to cross the artificial membrane and reaching the collection chamber.
Figure (7): Schematic representation of the T3SS purification hardware.
4. Dyeing
After expression and purification of our hagfish recombinant IF proteins, pre-dyeing of the proteins will be the next step in our project.
Our target is to successfully produce a myriad of colored fibres while maintaining the incredible tensile mechanical properties in hagfish IF. This will bring our products closer to a viable material for clothing, while at the same time reducing waste water and toxic chemical pollution generated from traditional dyeing processes.
Here we describe our dyeing system in 3 main areas
- Types of Dye
- Linkage of Dye
- Colours Variety
Each of these areas will be explored in detail in phase 2.
1. Types of Dye (Chromoproteins & Natural Dye)
Several criteria were considered when we decided which dyes to test:
Criteria of dye
- -Is it harmful or toxic?
- -Is it biodegradable?
- -Does it fade over time?
| Criteria | Descriptions/problem to solve/application in our project/ |
| Ability to biodegrade | A biodegradable dye would be the perfect match for a biodegradable fibre. |
| Toxicity | A toxic or harmful dye would not be suitable for everyday wear. |
| Resistance to fasting (fading due to oxidation) | A dye that loses its colors quickly would not be suitable for clothes, which are supposed to last long. We realise this criteria is in conflict with row 1 (ability to biodegrade), but we are trying to strike a balance. |
We chose chromoprotein to be our preferred dye IF fibres. One reason is that chromoproteins are encoded by a single gene only, which allows protein expression to be relatively easy compared to engineering the entire complex metabolic pathway of organic dyes. This simpler dyeing method introduces less variables to the experiments. Also, unlike fluorescent proteins, chromoproteins can produce vivid colours without a UV-light source. This also makes chromoprotein more suitable for application in dyeing clothes.
A total of 9 chromoproteins were selected and ordered from Twist Bio for our dyeing experiments. The basic information of these chromoproteins are summarized in the following table:
| Primary Colours | ||
| Red Chromoprotein | Blue Chromoprotein | Yellow Chromoprotein |
 |
 |
 |
| BBa_K592012 eforRed 25.6kDa Oligomeric State : Homo-tetramer Real photo  |
BBa_K864401 aeBlue 25.9 kDa Oligomeric State : Homo-tetramer Real photo 
|
BBa_K592010 amilGFP 25.9 kDa Oligomeric State : homo-tetramer Real photo 
|
| BBa_E1010 mRFP1 25.4 kDa Oligomeric State : Monomer Real photo 
|
BBa_K592009 amilCP 24.9kDa Oligomeric State : Tetramer Real photo 
|
BBa_K1943001 fwYellow 26.3 kDa Oligomeric State : Dimer Real photo 
|
| Secondary Colours | ||
| Purple Chromoprotein Red + Blue = Purple |
Green Chromoprotein Blue + Yellow = Green |
Orange Chromoprotein Red + Yellow = Orange |
| BBa_K mRFP violet mutant 25.3 kDa Oligomeric State : Monomer Real photo 
|
BBa_K592011 Cj Blue 26.1 kDa Oligomeric State : Homo-8-mer Real photo 
|
BBa_K1033913 scOrange 26.0 kDa Oligomeric State : Tetramer Real photo 
|
After consulting Dr. Fudge, we realized that using chromoproteins for dyeing could be problematic, as it may affect polymer formation when linked at the termini of the subunits. He suggested the use of dye with smaller molecular size : congo red, but after consideration, we decided not to use it because of its carcinogenic properties. Instead, we would use an alternative natural dye such as indigo. Natural dyes are chemical dyes with a smaller molecular size. It is hoped that it can bind to our IF without affecting polymerization of hagfish IF. We will be exploring more about this idea in phase 2.
We also reconsidered our choice of chromoproteins, placing stronger emphasis on smaller size and small oligomeric states because we would still try chromoproteins while minimizing the effect on IF polymerization.
2. Linkage of Dye (Covalently links to terminals & non-covalently binding)
To bind our chromoprotein to our IF, we first tried to covalently link the chromoproteins at different terminals in each subunit:
(1) Linking at N-terminal for both subunits (Alpha & Gamma)
(2) Linking at C-terminal for both subunits (Alpha & Gamma)
(3) Linking at N-terminal for Alpha subunits and Linking at C-terminal for Gamma subunits
(4) Linking at N-terminal for Gamma subunits and Linking at C-terminal for Alpha subunits
The thinking behind trying different combinations is to explore the effect of linking chromoprotein at different positions on fibres formations. This characterization could inform later teams, should they attempt to make fusion protein with hagfish IF subunits.
Figure (8). 2 cut sites are included in our genetic circuit: Acc65I is added at the N-terminal (between RBS and subunits) while AscI is added at the C-terminal (between subunits and his-tag). Both sites allow the insertion of chromoproteins, which are ordered in a separate fragment flanked by the same cut sites.
| Both subunits linking at N terminus | Both subunits linking at C terminus | Each subunit linking at different terminal |
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|
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However, a big concern for linking chromoprotein to IF covalently through peptide bonds is that this linkage may hinder the formation and self assembly of IF subunits into a polymer, and therefore adversely affecting the mechanical properties of our fibre.
Therefore, other methods for linking chromoprotein non-covalently will be explored in phase 2. By comparing various methods, we aim to optimize our dyeing system to produce high quality coloured fibres.
In the dry lab part, one 3D protein structure example of the fusion protein is being generated with the help of CHIMERA software. Both subunits (Alpha & Gamma) are linked with Blue chromoprotein aeBlue at N-terminals by the peptide bond.
In phase 2, we will be applying this model in VMD simulation to estimate the stability of chromoproteins linkage to our IF in different conditions, for example, testing its heat stability when heating to certain degree, which can provide us information on the optimal temperature of using the coloured IF and its tendency of colour fading when in high temperature.
3. Colours Variety (Using Distinct Colours & Colour Mixing)
After chromoproteins are integrated into the circuit to grant the threads vivid colours, Experiments to assemble subunits with different coloured chromoprotein will be performed to explore the possibility of colour mixing, thereby expanding our range of possible colours.
Our basic colour mixing system is to link different colours of chromoproteins in each subunit. Each subunit (Alpha & Gamma) is connected with chromoproteins at N-terminal and forms dimer, dimer will form tetramers and finally bound together as an entire intermediate filaments polymers. Thus by mixing in different ratios, we hope to create fibres with a variety of colours.
More theory supporting our colour mixing system will be illustrate in DRY LAB Page
5. Fibers Formations
Normally, our IF fibres should undergo a tensile test in the laboratory with real equipment to determine their tensile properties and accordingly propose the possible applications.
Under the COVID-19 pandemic, however, we could hardly produce the IF fibre themselves, not to mention carrying out an actual test on them. Hence, we turnt our focus on in silico modeling. We managed to predict the three-dimensional structure of hagfish slime intermediate filament dimer protein, and under the assumption that the dimer protein is the functional subunit of IF fibres, we tried to reveal the real-time conformational changes during draw-processing at the atomic level and also estimate the tensile properties of our product. In phase 2, we will try to construct the IF bundle polymer and demonstrate more accurate and detailed molecular basis of the outstanding tensile properties IF fibres possess.
References
[1] J. Lim, D S. Fudge, et al. “Hagfish slime ecomechanics: testing the gill-clogging hypothesis” The Journal of Experimental Biology 209, 702-710, Published by The Company of Biologists 2006
[2] D S Fudge, S Hillis, N Levy and J M Gosline. “Hagfish slime threads as biomimetic model for high performance protein fibres” Bioinsp. Biomim. 5 (2010) 035002 (8pp),IOP PUBLISHING
[3] T.Vehoff, A.Glišović, H.Schollmeyer, A.Zippelius, T.Salditt. “Mechanical Properties of Spider Dragline Silk: Humidity, Hysteresis, and Relaxation. Biophysical Journal Volume 96, Issue12, 15 December 2007, Pages 4425-4432
[4] L Cheng, X Tong, et al. “Natural Silkworm Cocoon Composites with High Strength and Stiffness Constructed in Confined Cocooning Space” Polymers (Basel) 2018 Nov; 10(11): 1214
[5] J. Pérez‐Rigueiro, C. Viney, J. Llorca, M. Elices. “Silkworm silk as an engineering material” Journal of Applied Polymer Science/ Volume 70, Issue 12
[6] V K Gupta. “Fundamentals of Natural Dyes and Its Application on Textile Substrates” Chemistry and Technology of Natural and Synthetic Dyes and Pigments,2019
[7] UCC Ireland. 2014. Sea DNA. iGEM wiki. https://2014.igem.org/Team:UCC_Ireland/Projects_SeaDNA.html
[8] F. Jing, G. A. Paul; P. Andrea, H. Nils, Lim, C. Teck; H. J. Matthew, M. Ali. 2017. Artificial hagfish protein fibers with ultra-high and tunable stiffness. doi:10.1039/c7nr02527k
[9] Fu, J. (2016). Biomimetic engineering of materials based on hagfish slime thread proteins. Doctoral thesis, Nanyang Technological University, Singapore.
[10] Jim Klein and Prasad Dhurjati. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1995. Protein Aggregation Kinetics in an Escherichia coli Strain Overexpressing a Salmonella typhimurium CheY Mutant Gene. 61(4).