Description


Introduction

Harnessing the biocompatible, strong and flexible nature of natural spider silk alongside the conductive and mechanical properties of reduced graphene oxide (rGO), our team aims to contribute to the field of tissue regeneration. By uniting these materials, our goal is to synthesise a biocompatible scaffold for use in biomedicine, more specifically tissue repair and subsequent injury rehabilitation.


The Problems

Problem 1: The prevalence of tissue damage and the struggle to treat it

Peripheral nerve injury has affected over 550,000 people in the last decade, and is becoming increasingly prevalent (Figure 1) [1]. Alongside 3-5 million tendon and ligament injuries occurring annually worldwide [2], global demand for regeneration therapies is widespread.

Frequency of Peripheral Nerve Injuries from 2009

Figure 1- Annual frequency of peripheral nerve injuries associated with sports and exercise-related peripheral nerve injuries[1]

Within both daily life and professional sport, nerve and connective tissues can be damaged to varying degrees of severity, impairing an individual’s mobility often for up to a year [1]. Current methods dominating the market of injury rehabilitation include physiotherapy and splints, but the only common internal support is grafts. Though promising, many postoperative complications (as well as compromising other tissues by extracting samples from them) remain with these therapies [3].


Problem 2: The harvesting of spider silk

Harvesting both silkworm and spider silk involves extraction from living animals, is labour intensive and presents pathogenicity risks [4]. Growing industrial demand for silk will simply not be met by low-yielding animal farms. Scaling up of spider farming specifically lacks potential as a viable solution specifically due to the territorial and sometimes cannibalistic nature of spiders [5].


Our Solutions

Problem 1: The prevalence of tissue damage and the struggle to treat it

For connective tissue rehabilitation, ex-vivo methods including kinesiology tape, splints and physiotherapy dominate the market; however their effectiveness is limited by the body’s healing rate. Autografts are the leading in-vivo method of ligament regeneration; providing integrated therapy, however, they require disruption to other tissues and can have issues with rejection [6]. Although evoking an immune response remains an unpredictable obstacle, our composite negates the need to compromise other tissues whilst also hosting many of the benefits of autografts.

Many researchers are focussing on conductive polymers for neuroregeneration, but a major drawback is that they are permanent, posing risk of long-term tissue damage or the inconvenience of multiple surgeries [7]. In theory, with our composite boasting biodegradability and conductivity, it has the potential to aid in faster and directed regeneration of nerves compared to other non-conductive counterparts without these long term complications [7, 8].


Problem 2: The harvesting of spider silk

Utilising Rosetta E. coli to synthesise our silk negates the need for animal exploitation via the potentially inhumane conditions that farmed spiders would be kept in. Furthermore, yield will be larger and more consistent compared to current methods of farming as bacteria can constantly produce proteins, whereas spiders don’t constantly produce silk.


Our Goals


Summary of our Goals

Figure 2 - Our three goals when designing the project.



Why take a synthetic biology approach?

After identifying these potential issues with the large-scale production of spidroin proteins from spider silk, we identified clear social, ethical, and industrial value in attempting a synbio-based alternative. This approach will enable us to provide more effective therapies for patients, reducing animal-components in industry and increasing the manufacture of a diverse yet scarce biomaterial. Other benefits include this synthetic silk being cleaner than that of silk farms, thus requiring less energy-costly stages of product purification.

Pairing genetic techniques with the modular nature of silk proteins presents the ability to tailor the properties of the silk to its application; elevating the potential effectiveness of therapies[9]. 76% of surgeons feel that using a tissue engineered anterior cruciate ligament (ACL) would be more appropriate than a patellar tendon, hamstring, or quadriceps autograft[10], demonstrating a clear demand for synthetic biology alternatives by stakeholders.


The Science

Mirroring its exceptional properties, spidroin is a notoriously challenging protein to produce synthetically [11]. This is due to its structure being abnormally rich in certain amino acids such as glycine and alanine[12]. Rosetta E. coli is our chassis for synthetic silk production as it overexpresses certain tRNAs that import these amino acids in high demand[12]. Additional adjustments were made to try to maximise our yield; namely sequence encoding CycA and TrxA. CycA, is a permease that is involved in transport across the cytoplasmic membrane of D-alanine [13], hoping to increase intracellular alanine. Furthermore, we encoded a thioredoxin protein followed by a thrombin cleavage site to increase solutbility [8]. MaSp proteins should precipitate out of solution and spontaneously assemble into fibres upon cleavage of this site[8].

Adhering rGO to silk has shown to be successful in enhancing properties such as conductivity whilst not significantly hindering mechanical ones[14]. Liang et al’s[15] method whereby silk fibres were mixed in an rGO suspension and filtered formed the basis of our rGO binding protocol, but this was later adapted. PVA and butanediol were later used to try to increase Young’s modulus and flexibility of the eventual film. Both have good biocompatibility[16, 17].


Our Inspiration

The potential of spider silk piqued our curiosity after watching a BBC 2 Horizon documentary about synthetic biology entitled “Playing God” [18]. Dr Adam Rutherford, British geneticist and former editor of the journal ‘Nature’, visited a goat farm that housed genetically engineered goats to produce spider silk in their milk[18]. During the programme, Professor Randy Lewis of Utah State University highlighted a demand for silk, particularly spidroin, due to its exceptional tensile strength properties. This gives it potential in sectors spanning textiles, space materials and biomedical engineering.

Given the episode was first aired a decade ago, we were intrigued if and how experiments involving spider silk have been developed further. While substantial research has already been performed in terms of modifying the properties and production of silk, additions such as composite formation have not yet been extensively expanded. One of such novel aspects we identified is attempting to bind rGO to silk to enhance its conductivity, broadening its relevance in regenerative medicine.

Within iGEM, we were inspired by two teams: UCLA 2015[19] and GreatBay SZ 2019[20]. UCLA aimed to address issues that come with producing synthetic silk; the problem being that natural silk contains many highly repetitive and conserved coding regions, which E. coli cell machinery struggles to process. To combat this, the team attempted to modularize the regions involved in silk production so the proteins would be produced more consistently. Directed assembly was then used so that the proteins would assemble into a specific sequence. We plan on using a similar technique involving modularity and directed assembly to construct our modified silk. When it came to plasmid design, we took inspiration from the BioBrick contribution of the 2019 GreatBay SZ team. Similar to UCLA, they worked to improve the production of synthetic spider silk but focussed on ease of manufacture and environmental friendliness. Our projects share these values and we have used some of their BioBricks as credited in our parts collection.


Why this project?

At the end of our “bootcamp week”, a team building introduction to all things SynBio as organised by our PIs, we went straight into idea brainstorming. After toying with many project possibilities, we landed on three main contenders: cadmium recycling, artificial antibody production and a spider silk-rGO composite. We split into small groups for more detailed research into previous advances and the promise of each idea and then presented them to academics at the university. Questioning from and discussions with these academics helped us to more critically reflect on our work and identify the unique logistical challenges that each project presented.


Feedback from this was largely in favour of our final choice as it seemed to host the most everyday benefits whilst also being easier to checkpoint our successes. We concluded that biomedical applications were the most pressing as there is already a large awareness and movement to improve the recycling industry and antibodies are mainly beneficial from a scientific lens only.

Given that we have our own Graphene Centre here in Exeter, we organised a meeting with Dr Ana Neves, a senior Lecturer in Materials Engineering who specialises in the study of graphene for flexible and wearable applications. Dr Neves welcomed the idea, increasing our optimism about the project’s success given that someone with such expertise considered this composite to be an achievable goal.

Compounding all of this feedback, we had a group discussion which ended with an anonymous vote for our favourite project. The result was unanimously in favour of making a synthetic silk-rGO composite. The foundations of BionExe were set!


References

  1. Li NY, Onor GI, Lemme NJ, Gil JA. Epidemiology of Peripheral Nerve Injuries in Sports, Exercise, and Recreation in the United States, 2009 – 2018. 2021. The Physician and Sportsmedicine. 2021 Jul 03;49(3):355-362. Available at https://doi.org/10.1080/00913847.2020.1850151M
  2. Bullough R, Finnigan T, Kay A, Maffulli N, Forsyth NR. Tendon repair through stem cell intervention: cellular and molecular approaches. Disabil Rehabil. 2008;30(20-22):1746-51. https://doi: 10.1080/09638280701788258.
  3. Sun J, Wei XC, Li L, Cao XM, Li K, Guo L, et al. Autografts vs Synthetics for Cruciate Ligament Reconstruction: A Systematic Review and Meta-Analysis. Orthop Surg. 2020 Apr;12(2):378-387.https://doi.org/10.1111/os.12662.
  4. Zhang Q, Li M, Hu W, Wang X, Hu J. Spidroin-Based Biomaterials in Tissue Engineering: General Approaches and Potential Stem Cell Therapies. Stem Cells Int. 2021 Dec 20;2021:7141550.https://doi.org/10.1155/2021/7141550.
  5. Wise DH. Cannibalism, food limitation, intraspecific competition, and the regulation of spider populations. Annu. Rev. Entomol.. 2006 Jan 7;51:441-65.
  6. Longo U, Rathbone S, Maffulli N, Cartmell SH. Most British Surgeons Would Consider Using a Tissue-Engineered Anterior Cruciate Ligament: A Questionnaire Study. Stem Cells International. 2012 Feb 26. Available at https://doi.org/10.1155/2012/303724
  7. Schacht K, Scheibel T. Processing of recombinant spider silk proteins into tailor-made materials for biomaterials applications. Curr Opin Biotechnol. 2014 Oct;29:62-9. https://doi.org/10.1016/j.copbio.2014.02.015.
  8. Rasooli F, Hashemi A. Efficient expression of EpEX in the cytoplasm of Escherichia coli using thioredoxin fusion protein. Research in Pharmaceutical Sciences. 2019 Dec;14(6):554.
  9. Hinman MB, Jones JA, Lewis RV. Synthetic spider silk: a modular fiber. Trends Biotechnol. 2000 Sep;18(9):374-9. https://doi.org/10.1016/s0167-7799(00)01481-5.
  10. Mayo Clinic. ACL reconstruction [internet]. Mayo Clinic; 2020. [updated 2021 Mar 6; cited 2022 Sept 15]. Available at https://www.mayoclinic.org/tests-procedures/acl-reconstruction/about/pac-20384598#:~:text=Within%20the%20first%20-few%20weeks,can%20return%20to%20their%20sports.
  11. Chung H, Kim YT, Lee YS. Recent advances in production of recombinant spider silk proteins. Current Opinion in Biotechnology. 2012 Dec 01;23(6):957-964. Available at https://www.sciencedirect.com/science/article/pi-i/S0958166912000626
  12. Tegel H, Tourle S, Ottosson J, Persson A. Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3). Protein Expression and Purification. 2010 Feb 01;69(2):159-167. Available at https://www.sciencedirect.com/science/article/pi-i/S1046592809002150.
  13. Hook C, Eremina N, Zaytsev P, Varlamova D, Stoynova N. The Escherichia coli Amino Acid Uptake Protein CycA: Regulation of Its Synthesis and Practical Application in l-Isoleucine Production. Microorganisms. 2022 Mar 27;10. Available at https://www.mdpi.com/2076-2607/10/3/647.
  14. Kiseleva AP, Krivoshapkin PV, Krivoshapkina EF. Recent advances in development of functional spider silk-based hybrid materials. Frontiers in Chemistry. 2020 Jun 30;8:554.
  15. Liang B, Fang L, Hu Y, Yang G, Zhu Q, Ye X. Fabrication and application of flexible graphene silk composite film electrodes decorated with spiky Pt nanospheres. Nanoscale. 2014;6(8):4264-74.
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  20. Great Bay SZ. ‘SPIDERMAN: Spidorin engineering with chromoprotein and natural dye’ iGEM Foundation. 2019. https://2019.igem.org/Team:GreatBay_SZ.

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