Team:HKUST/Implementation

• Project
  • Description
  • Design
  • Contribution
  • Improvements
  • Safety
• Dry Lab
  • Protein Structure Pretiction
  • Molecular Dynamics Simulation
  • Color Mixing
• Wet Lab
  • Description
  • Cloning
  • Expression & Purification
  • Thread Formation
  • Color Mixing
  • Draw-Processing
• Human Practice
  • Implementation
  • Integrated Human Practice
  • Collaborations
  • Education
  • Partnership
  • Entrepreneurship
• Team
  • Team Members
  • Attributions
• Parts
  • Parts Overview
  • Basic Parts
  • Composite Parts
• Awards
  • Award Criteria
  • Achievements

Implementation

• Proposed Implementation

• Proposed End Users

• Our Vision

• Implementation in the Real World

• Safety Considerations

• Risks and Challenges

Proposed Implementation

Previous efforts in synthesising recombinant hagfish fibres have yielded fibers of mediocre strength and quantity [1] [2]. 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 clothing industry.

Our mass production system is comprised of five (5) aspects:

Using our mass production system, we hope we could produce fibers from hagfish Intermediate Filament while maintaining fiber quality and fiber yield. With chromoproteins as the dyeing method, we could mix chromoproteins in a certain ratio where fibers of different colours are produced. We hope we could provide coloured fibers for textile companies for knitting our fibers into the production of clothes.

Proposed End Users

The proposed end users are textile manufacturers, designers who make use of our material, and customers who buy products made from our material. Because our product will be used by different users in different ways, we have contacted some of them for opinion and feedback so that we may better satisfy their needs.

For example, Esqual Group has invited us to visit their manufacturing sites in Mainland China so that we may see in person the manufacturing process of clothes.

From our survey results, most respondents are willing to adopt a more sustainable clothing habit, as reflected in their willingness to pay more for a greener t-shirt. This tells us there are at least some potential customers in the area who would buy clothes made from IF threads.

Our Vision

Since hagfish intermediate filaments (IF) have been shown to possess incredible mechanical properties, we imagine threads made from the IF could be used to make high-performance sportswear, bandages, biodegradable fishnets, and the like. It has many of the same properties as spider silk and therefore could be an alternative that is easier to produce.

Implementation in the Real World

We hope to manufacture a new kind of textile material from hagfish intermediate filaments (IF). Additionally, we also want to fuse the IF proteins with chromoproteins to give them color. Through mixing different chromoproteins linked to IF, we may alleviate the reliance on highly polluting dyes.

In practice, this involves creating a production method that can express, purify, and polymerize IF proteins beyond the lab scale. This has been a challenge, because previous attempts at synthesizing recombinant IF are focused at characterizing its mechanical properties. However, we have proposed some modifications to the circuit and the method of expression which may boost the product yield.

For example, we reasoned that protein expression rates could be adjusted, firstly, by using different constituent promoters (T7 promoter and K88 promoter) to increase yield, and secondly by using signal peptides for an alternative purification method.

Safety Considerations

The toxicity of a dye is an important factor when we determine whether a potential dye is suitable for our project. For example, in our interview with Dr. Fudge, he suggested the use of dye with smaller molecular size: congo red. However, after consideration, we decided not to use it because of its carcinogenic properties. Instead, we would use an alternative natural dye such as indigo.

The injectisome system in Synthetic Injector E. coli (SIEC) we planned to use for implementing the novel purification method next phase originates from Enteropathogenic Escherichia E. coli (EPEC). EPEC can cause diarrhea in humans when adhered to intestinal tissue and successfully injects effector proteins via its type 3 secretion system (T3SS). SIEC possess the following characteristics that fulfill the safety requirements:

• - Non-pathogenic chassis E. coli K-12
• - Inducible assembly of T3SS filamentous injectisome
• - Removed effector genes and its related regulators

As the effector proteins are removed, the strain is deemed non-pathogenic and thus belongs to biosafety risk group 1. By choosing this particular bacterial strain, we hope to minimize the potential risk when working with it next phase.

Risks and Challenges

The biggest technical challenge at the moment is scaling up production. We have looked into possible methods to produce large amounts of intermediate filament proteins continuously and preferably autonomously. Currently we are looking into the T3SS secretion mechanism as modified by UCopenhagen 2018 and have proposed ways we can implement their secretion system. The two major steps in such an implementation is outlined below:

• - 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.
• - Transforming our chassis with T3SS vector: A T3SS plasmid would also have to be transformed apart from the plasmid containing our IF circuit. Double-transformed cells would be adsorbed on an artificial membrane via the action of the injectisome, which is encoded by the T3SS genes transformed in the bacterial genome. The injectisome would then penetrate the artificial membrane. This allows our IF proteins tagged with the T3SS signal peptide to cross the two membranes and ultimately be collected in the phosphate buffer on the other side of the artificial membrane.

Aside from technical challenges, how to convince people to try out our novel material is also a challenge. The results from our consumer behavior survey shows that the majority of respondents are relatively passive consumers who change buying behaviors slowly. Therefore, we should focus on the younger generations who are more accepting of new products and gene editing technologies.

References

[1] UCC Ireland. 2014. Sea DNA. iGEM wiki. https://2014.igem.org/Team:UCC_Ireland/Projects_SeaDNA.html

[2] 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