Further Research

Project Improvement:

The realisation of E.lectrode for real-world benefit would require further steps for complete characterisation of the new modified pili. To finalise our study, we would need to determine whether GspilA is still expressed when new collagen tags are added to the monomer. For this, testing for protein production of PilA monomer with a His tag would be required. Even though Ueki et al. (2019) has determined it is possible to produce His-tagged PilA, our new 2-plasmid approach to Type IV pili machinery expression in E.coli would require us to perform anti-6HIS and anti-PilA western blot analysis to confirm monomer expression. We have also explored the possibility of performing an ELISA to quantify collagen binding of GsPilA with our new collagen tags. After protein purification of the new skin-binding pili (Protein Purification Lab Book), atomic force microscopy would also be a viable route for confirmation of protein expression and successful purifcation [1]. It would also be necessary to delete all fimbrial and type I pili-associated genes to ensure maximum success for the purification step of our procedure. Successful completion of these delineated steps would:

• 1)Confirm heterologous expression of GspilA with novel binding tags is possible. (and all its implications regarding conductivity and purification yield)
• 2)Confirm genes of pathogenic EHEC E.coli, avoided by the team for safety concerns (See Safety) are not required for pili production.
• 3) Further characterise binding ability of GsPilA with new skin-binding tags.

We would also be required to test for conductivity of GsPilA-TKKTLRT and GsPilA-LRELHLNNN as the addition of the tag may have disrupted the structural aspects of Geobacter sulfurreducens pili that confer it with its characteristic conductivity. The team has drafted a new and simplified protocol for conductivity testing (See Conductivity Protocol). For industrial production of GsPilA with new skin-binding tags it would be appropriate to evaluate new chassis options across strains of E.coli that yield a superior amount of Type IV pili. Szmuch et al. (2023) [2] have successfully expressed, purified and characterised novel metal-binding Geobacter pili in Shewanella oneidensis, a species that expresses its own type IV pili in liquid culture. Throughout the project, there was recurrent debate on the possibility of modifying E. coli strains with type IV pili assembly machinery genes that are not cryptic as in E. coli K12. [3]

Alternative Skin-Binding Tags:

After successfully synthesizing pili with collagen tags, future research can build on this proof of concept by expanding the scope of our approach and engineer pili with additional skin-binding tags that bind ceramides. Ceramides are essential lipids located on the surface of the skin, where they form a key component of the epidermal barrier. They play a crucial role in retaining moisture and protecting the skin from external environmental stressors [31]. The method for binding to ceramides would closely resemble the approach that was used for collagen binding. Collagen-binding tags were successfully attached to the C-terminus of the nanowires to facilitate skin binding. While proteins typically do not bind directly to ceramides, there are two ceramide-binding tags that can be used: TKVTG SLETK YRWTE YGLTF TEKWN TDNTL GTEIT VEDQ and GKVTG TLETK YKWC EYG LTF TEKWN TDNTL GTEIA IEDQ. These binding sequences are derived from voltage-dependent anion channels VDAC1 and VDAC2, which serve as mitochondrial ceramide-binding proteins.[4]

We have also evaluated the use of longer skin-binding peptides used in the cosmetic industry, specifically the chondroitin sulfate skin-surface binding peptide, which is a 15-amino acid motif located upstream (RKQRRERTTFTRAQL, RK-peptide) of the Otx2 homeodomain. [5] We also considered the use of keratin-binding peptides such as the one described by the Manchester 2019 iGEM team BBa_K2906100. The presence of keratin in the skin and the short length of this previously characterised peptide makes it a potential candidate for our future research plans.

Spider-Silk Sleeve:

When debating the final product appearance of our skin-binding GsPilA, E.lectrode also considered making a spider silk-pili hybrid that could be used as a fabric material for the sleeve in prosthetics worn around the residual limb. This aims to address the discomfort issues with prosthetic sockets that were highlighted during our stakeholder meetings. (See Human Practices ). The sleeve would be intrinsically conductive and capture sEMG (Surface Electromyography) signals directly from the skin serving as an intermediate between the electrode and the skin. When researching potential ways to achieve this outcome, we came across the UCopenhagen 2022 iGEM project which developed a Tag catcher system that can potentially attach our modified pili monomers to the spider silk protein. In a final version of the project, the modified pili could be embedded in a spider silk sleeve and as so be integrated in a composite material while still binding and capturing signals from the skin surface. Currently the SnoopCatcher (BBa_K4247009) sequence is too long for addition to GsPilA monomers and as so E.lectrode would have to explore larger conductive pili that can express longer tags (See Model). This dual binding system would also require expression of two different peptides as in Ueki et al. (2019) [6].

Integration

Prototype Brainstorming Timeline

As our project advanced in terms of design and production of modified pili, so did our concept of what a final product would look like. The team tried to keep a broad perspective and imagine how the nanowires would be applied in a prosthetic device. Initially, the team conceived protein nanowires encased in a spider silk coating. At this time, the team name that was being discussed was “Silk-E-Pili” and the perspective was one of pili as insulated conductive wires that could serve as both sensor and maintain a single point of contact with the receptor. This “plug and socket” approach proved valuable at the simpler stages of the project but slowly fell out of use as we further got to know about how prosthetics and e-pili function.

As the project developed, the team went through many stages of how protein-based conductive nanowires would look like in a prosthetic. It was even debated whether the format of our ideal prototype should be in the form of a cream that is applied on top of the skin that creates a conductive barrier between skin, and an electrode which would be taped on top it. Our meetings with the British Association of Prosthetics and Orthotics and the University of Salford’s Prosthetics and Orthotics Department provided us with the unique opportunity of meeting experts in this research field. Their comments and critique of our project forced us to rethink and perform several iterations to our project’s design. Our main takeaways from both meetings can be found at the Human Practices page Our final project concept consists of a nanowire coating for the electrodes in prosthetics as shown in Project Description.We have also explored the possibility of integrating the protein nanowires in a spider silk e-pili composite sleeve for the prosthetic socket.

Other Human Machine Interfances and Current Research: Wearable Devices and Electric Skin

Considering the current research focus of E-pili for energy generation and the development of genetically tailored G. sulfurreducens e-pili that can function as the sensing component in electronic sensors (because their conductivity changes in response to interaction with diverse analytes), there is potential for implementation of skin-binding e-pili in self-powered biomedical sensing wearable devices and electronic skin [7].

Skin-binding electrically conductive pili that are also designed with the ability for electrode binding [8] could be fused with current advancements in in situ tethering of organic bioelectronic fibres that take inspiration from spider silk [9].

Geobacter nanowirefilms have been shown to generate power from ambient humidity and maintain a stable direct-current voltage of some 0.4–0.6 V for more than 2 months; a voltage of 10 V was achieved by connecting 17 devices in series and the connected devices were able to power a LED and LCD. A single device could power a semiconductor nanowire transistor for logic operation which could lead to sustainable and compact computing systems [11].

There is also ongoing research funded and incubated by the European Commission's ‘Worth Partnership Project’ to develop an electric skin biomaterial based on calcium alginate composites with silk fibroin, chitosan and glycerin as plasticiser which is then combined with G. sulfurreducens pili nanowires. The aim of the project is to power electronic devices with ambient air by providing them with a “skin” that can serve as a flexible battery adaptable to many forms of technology. One of the material parameters denoted on the Electric Skin’s toolkit for open community research [12] is the current struggle with nanowires and conductive material to stay adhered over time. The research group’s focus has been plasma etching and its potential to tune the biopolymer’s surface adherence but our approach and proof of concept offers an alternative for adherence to skin.

There is also extensive research on the potential applications of Geobacter e-pili for bioremediation. There is ongoing research on the ability of Geobacter species to detoxify heavy metals and radioactive elements through pili-mediated reduction [14]. The use of Geobacter pili as memristors to emulate biological memory and record biological electrical events of low voltage has also been explored as a way of developing sensor-driven, integrated neuromorphic interfaces for “bio-emulated interfaces and microsystems” (Fu et al., 2021) [15].

Pre-Clinical and Clinical Trials

Testing for Biocompatibility

As part of our new basic part additions to the iGEM Standard Parts registry, the team researched published reports of allergenicity testing for the GspilA with both collagen-binding tags (See Our New Basic Parts) but further research should be conducted. After our meeting with the Manchester Institute of Innovation Research raised concerns over pili-derived molecules or nanowires and their ability to trigger immune responses we did further research to understand how we can ensure biocompatibility for our final product. (See Human Practices) Once synthesised, pili with skin-binding tags will need to undergo rigorous testing for biocompatibility, to ensure they can integrate safely and effectively with biological tissues. This step is critical for advancing the application of this technology in medical or therapeutic contexts. ISO 10993 are standard guidelines for assessing biocompatibility of all medical devices that encounter human tissue. Therefore, these guidelines must be used for assessing biocompatibility of our nanowires. According to ISO 10993 guidelines our skin-specific nanowires fall under the intact skin surface device category with a contact duration of less than 24h up to 30d, which is classified as a “prolonged exposure”. Key considerations under this classification include:

• Cytotoxicity: The device must be tested for any potential toxic effects on cells, by in-vitro extracting potential toxins from the medical device using cell culture methods, as outlined by ISO 10993-12. The resulting extracts are exposed to mammalian cells derived from mice, such as L929 or BALB/3T3 cells, at varying concentrations. Cytotoxic effects are evaluated through both qualitative tests, like colony formation, and quantitative assays, including NRU, MTT, and XTT [16]
• Sensitization: Evaluation of whether the device could cause allergic reactions after prolonged contact.
• Irritation or Intracutaneous Reactivity: Testing for local irritation to tissues or skin.
• Systemic Toxicity: Assessment of any adverse effects on organs or biological systems as a result of the device's components.
• Subacute and Subchronic Toxicity: Since the device will be in contact with the body for up to 30 days, testing for potential toxicity over that period is required.
• Material Characterization: Analysis of the materials used in the device to ensure their safety for prolonged use. [17]

Phase 2 - Virtual Testing

One of the key clinical trials we plan to conduct is a usability test, where our product will be evaluated by having representative users interact with it in real-world scenarios. The main objective of this test is to uncover any usability issues and gather valuable feedback that can be used to refine and improve the overall user experience [18]. This process involves carefully observing users as they perform specific tasks, offering valuable insights that highlight areas for potential improvement. The first stage of such trial would be adapted from Yu et al. (2021) [19]. It would consist of fourvirtual training sessions of one hour, where participants would be trained with the Virtual Integration Environment (VIE) for upper-limb prosthetic training (an open-source version of the code is available at https://bitbucket.org/rarmiger/minivie). This would help capture sEMG signals from the participants’ residual limbs and filter them using signal analysis algorithms. Participants would be required to complete a 1-DOF Target Achievement Control (TAC) test to characterise the accuracy at which motion classes were achieved. [14] For the described protocol, an average TAC score ≥ 75% was needed to qualify for Phase II. To assess the performance of our nanowires and identify any potential issues, a group of upper limb prosthetic users would be recruited. Prosthetics integrated with our nanowires would be provided to these participants, and feedback would be collected regarding the precision of movement and overall comfort of the prosthetic, particularly in comparison to their previous socket. After conducting the usability test and the first stage of the Yu et al. (2021) protocol, the data and feedback must be thoroughly analyzed, as this will be instrumental in determining the effectiveness of the technology and guiding future developments of the product.

Phase 3 - Testing in Patients

For the participants that qualify for the second stage of our trial, electrode number and orientation would need to be adapted according to amputation level and residual limb anatomy. “The first (i.e. baseline), sixth (i.e. midpoint), and 12th (i.e. exit) sessions would include functional and subjective testing which would include the Box and Blocks (BB) [20] test, Jebsen-Taylor Hand Function Test (JHFT) [21], and Assessment of Capacity for Myoelectric Control (ACMC) [22]“ [19] “The BB test pertains to efficiency in repeating a motion and grasp sequence to transfer blocks over a raised barrier. The JHFT evaluates the ability to perform seven activities of daily living. The number of objects moved per second is calculated to track improvements in device control. The ACMC assesses the effectiveness of myoelectric device use during activities of daily living—dressing, food preparation, ironing, packing a suitcase, or driving—and was developed and validated for upper-limb prosthetic outcomes testing.” [23]

Marketing and Commercialisation

Regulation

In the United Kingdom, external prosthetic devices and orthotics manufacturers should also be registered as a Class I manufacturer and meet the definition of a medical device on Medical Device Regulations 2002 (SI 2002 No 618, as amended) [24] and follow the requirements for the device on Part II of the UK MDR 2002, Annex I. [25] There is also certain legislation considerations that any synthetic biology invention has to abide to [26]:

• Health and Safety at Work Act 1974
• Control of Substances Hazardous to Health Regulations 2002
• Genetically Modified Organisms (Contained Use) Regulations 2014
• The National Security and Investment Act 2021 (Mandatory notification to the government for companies carrying out activities in relation to “advanced materials”)
• The Anti-Terrorism, Crime and Security Act 2001 (Contains measures to prevent misuse of pathogens / toxins
• The Patents Act 1977 (and its criteria)
• Human Medicines Regulations 2012
• Environmental Protection Act 1990 and the Environment Act 2021

In terms of regulatory approval, as our project entails a non-invasive sEMG prosthetic, it would be classified as a Class 2 rather than Class 3 medical device, this would reduce FDA average approval time by 100 to 182 days when compared with other prosthetic types such as implanted prosthetics. In the UK approval of medical devices is under the designation of Medicines and Healthcare products Regulatory Agency (MHRA). MHRA processing times typically consist of around 90 days.

Patenting

We have identified the following patents and patent applications related to Geobacter sulfurreducens microbial nanowires:

• WO 2020/191281 A1 “Microbial Nanowires Modified to Contain Peptides and Methods of Making” : Derek R. Lovley (Amherst), Toshiyuki Ueki (Amherst, MA) World Intellectual Property Organization Published 24 September 2020 US Patent 9,017,720: "Methods for improved production of electricity in microbial fuel cells using conductive pili and genetically modified microorganisms."
• US Patent 8,569,443: "Bacteria and methods for production of electricity."
• US Patent 10,469,775: titled "Electricity-Generating Protein Nanowires for the Direct Extraction of Energy from Air".
• US Patent 9,601,227: “Microbial nanowires and methods of making and using”

As such, we have studied and evaluated alternative electrically-conductive pili that can be modified with skin-binding peptides as part of our Dry Lab. Monomers of over 100 amino acids of other e-pili with a N-terminal region homologous to G. sulfurreducens may also offer the advantage of heterologous expression in E. coli of monomers with longer skin-binding peptides. [29]

Early Market Engagement

Once a final iteration of the product has been reached, our previous and very informative meetings with experts in prosthetics should be built upon with further stakeholder engagement. In particular, the team would require support with commercialisation, and should seek constant contact with current prosthetics suppliers. The University of Manchester Innovation Factory is responsible for the commercialisation of intellectual property (IP) developed at the University of Manchester. It would be in the project’s best interest to make use of this available resource and their extensive expertise. The Innovation Factory’s process consists of 5 stages to ensure development of a commercialisation plan, define the value of the opportunity and protect IP. This process and the one for pre-filling a patenting application are shown below:

E.lectrode would also prioritise becoming a part of “Sister” a new 4 million ft² innovation district specialising in the advancement of digital tech, health innovation, biotechnology, advanced materials and manufacturing and connecting early-stage high growth potential businesses with investors [34]. E.lectrode would aim to partner with the Industrial Biotechnology Innovation Catalyst (IBIC), one of the four catalysts that will be based in the new innovation district to become one of the 25 early-stage companies supported to scale-up IBIC aims to achieve by 2028. We also came across a Manchester-based private prosthetics service, Beast Prosthetics, which specialises in offering patients access to innovative prosthetic solutions. By approaching this company and others across private healthcare and the NHS (such as Steeper Group, STEPS Prosthetics, Koalaa, Ottobock, Swift Prosthetics, Ossur and clinicians), we aim to break into the market and ensure our product can be safely delivered to patients. We would also aim to maintain the human practices reflexivity criteria that iGEM educates teams on, and iterate the product according to the needs, worries and reviews left to us by the prosthetics’ users. This would be performed in similar fashion to the DBTL cycle work methodology we performed for our activities throughout the entirety of the project. (Check our DBTL Cycles in the Engineering page) We understand there may be ethical concerns regarding the marketing of our prosthetic medical device to clinicians. As so, we would adopt the measures proposed by Keller et al. (2016) [30] designed for the relationship between a physician and the pharmaceutical industry. Most importantly, E.lectrode would require any clinician to declare any conflict of interest, immediately upon their initial approach with patients. The 4Ps of marketing (Product, Price, Place, Promotion) have been taken into consideration throughout the project. As previously mentioned, the place (or method of delivery to patients) would be through specialised clinicians and promotion would take on strict guidelines to ensure patients are the designated priority. Evaluating our product, potential customers and what is the innovative aspect of our project was part of our human practices approach to iGEM when meeting with essential stakeholders in the prosthetics industry (See Human Practices). Price could only be defined at a later stage of the project and would depend on ensuring sufficient scale-up of the pili yield in E. coli or other adequate chassis, optimizing production and the cost of efficient quality control to guarantee patient safety.

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