Design

E.lectrode: Connecting technological interface with human expression through recombinant proteins

From early human cave art to operating a pipette, hands are an integral part of the human identity and expression that allows people to connect with others.

Cave Art in Cueva de Las Manos shows hand prints
Figure 1: Cave Art in the Cueva De Los Manos in Argentina [1]. This illustrates the importance of hands in human expression since the beginning of time.

With the global prevalence of amputation, what if this integral part of human identity was compromised? Myoelectric prosthetics are a popular choice to substitute for these lost capabilities [2]. There are two different types of myoelectric prosthetics that are commonly found on the market: Multi-Grip and Single Grip [3]. Multi-grip prosthetics give a wider range of motion because fingers can be moved separately(multiple articulations), while single grip usually has a limited range of motion.

Types of prosthetics: Single Grip and Multi-Grip
Figure 2: Different types of prosthetics [4]. Single Grip and Multi-grip prosthetics.

However, one of the major problems of these prosthetics is the lack of sensory feedback [5]. Modern prosthetics can suffer from delays in movement caused by uneven signal transmission due to motion artefacts and electrode movement, lowering user experience and usability of prosthetics [5]. This lack of feedback can cause frustration and prosthetic abandonment, which can both directly and indirectly lead to more than $1 million USD of health costs for amputees before accounting for any loss of wages or salary due to an inability to work [6].

Problems with Myoelectric Prosthetics leads to abandonment
Figure 3: Types of myoelectric prosthetics. There are 2 types of myoelectric prosthetics with different ranges of motion, but both of them have similar issues where patients don’t get enough benefit from the prosthetic leading to prosthetic abandonment [3].
F

E.lectrode seeks to improve myoelectric prosthetics’ electrode-skin interface and create enhanced interpretation of electrical impulses in order to facilitate patient autonomy and help them reduce the movement lag of prosthetic limbs.

Introducing our Synbio Solution

E.lectrode aims to engineer the non-virulent bacteria Escherichia coli NEB 5-alpha to express electrically conductive type IV pili (E-pili). These pili are derived from Geobacter sulfurreducens, with an additional protein-binding tag at the pili’s carboxyl end to create a recombinant protein [7]. This creates protein nanowires that adhere to skin to reduce motion artefacts and fix the electrode to its contact point on the residual limb.

Geobacter sulfurreducens and type IV pili

Pili are hair-like protein appendages found outside of the membrane of numerous bacterial species [8]. They serve a wide range of functions, including locomotion, skin adhesion or conjugation [9]. Geobacter sulfurreducens, an anaerobic gram negative bacterium found in soil, uses its pili for electron transport [10]. This allows the bacterium to reduce metals, such as iron [11]. The high number of aromatic residues are thought to facilitate this function, where the side chains interact by pi-pi stacking and allow electrons to jump from one aromatic ring to the next [12].

Type 4 pili expression machinery
Figure 4. Type 4 pili expression machinery. Each letter corresponds to one of the components; B - hofB, C - hofC, D - the pilin subunit, M - hofM, N - hofN, O (purple) - hofO, O (yellow) - gspO, P - hofP, Q - hofQ [13].

Structurally, the pili are polymeric in nature, formed from a number of subunits called pilins [15]. Pilis are assembled by a complex assembly machinery that spans the bacterial membrane. The assembly machinery is composed of a multitude of protein units that serve various functions (Table 1).

Table 1. Proteins involved in the Pilin assembly machinery complex
Protein Unit Function
HofB ATPase
HofC Platform protein
HofM Assembly protein
HofN Assembly protein
HofO Assembly protein
HofP Assembly protein
HofQ Assembly protein
PpdA Minor pilin
PpdB Minor pilin
YgdB Minor pilin
PpdC Minor pilin
GspO Prepilin peptidase

Why Collagen?

Collagen is used as an initial proof-of-concept target for our protein binding pili. It is the most prevalent fibrous structural family of protein [16] in humans comprising around 30% of the human body's dry weight [17]. It is found in skin, tendons, bones, cartilage, and all other connective tissue. Apart from its contribution to the structural strength of biological tissues [18], collagen is a signalling moleculs that regulates several intrinsic and extrinsic pathways [19]. Although there are at least 16 different types of collagen in the human body, types I, II, and III are the most common [17]. Out of these, type I comprises over 90% of the collagen in the human body [20].

Apart from collagen’s abundance in the body, its binding tag proved a suitable choice for proof of concept experiments due to its well characterised nature [21]. Furthermore, it is practical to test in the lab due to its ease of procurement and cost effectiveness.

There is a plethora of literature exploring different binding tags to collagen which makes it a favourable option to explore [21]. This led to utilising collagen binding tags from Decorin (LRELHLNNN) and Collagenase (TKKTLRT) [22] which fall under the optimal amino acid range for tag selection and attachment to pili complex [23]. Decorin promotes controlled release of molecules and collagenase promotes vascularization and cellularization [24].

Collagen binding is intended as a proof of concept for the project. E.lectrode ultimately aims to create pili that bind to common skin-surface molecules for its final product [25].

The E.lectrode Approach

The engineering of biology to solve real world issues has been at the heart of iGEM from the beginning. To emulate this problem solving aspect of engineering as the core of the project, the design thinking cycle was integrated into the E.lectrode approach. Unlike the traditional engineering cycles that are often solely experimentally focussed, our approach takes into account initial research on the need and demand of our problem. It aims to scope out the gap in the market and define problems to further guide scientific advancement and lab work. The benefit of this approach is that it takes into account real world problems and designs user-centric solutions. Thus, it bridges the gap between scientific innovation and practical use.

E.lectrode Approach Cycle: 1) Empathise, 2) Ideate, 3) Prototype, 4) Test, 5) Implement
Figure 5. The E.lectrode Approach. This cycle is adapted from the design thinking cycle which guided the entire project. The stages entail: stage 1: Empathise—Conduct research to understand the users' needs. Stage 2: Ideate—Question existing assumptions and generate new ideas. Stage 3: Prototype—Begin developing potential solutions. Stage 4: Test—Evaluate the solutions by putting them into practice. Stage 5: Implement—Exploring future outlook. The cyclical diagram illustrates the iterative process and non-linear capabilities of the approach that allows to refine the project.

1. Empathise

As a way to gain understanding of the scope of our issue and demands that our project needed to address in the field of prosthetics, different key persons' feedback ranging from researchers to potential end users was integrated into the project. There were various challenges associated with prosthetics explored by engaging with clinicians and researchers.

To enrich the project approach, broaden insights and design effectively, three key institutions were consulted. This included Manchester Institute of Innovation Research [26], British Association of Prosthetics and Orthotics (BAPO) [27] and the University of Salford’s Prosthetics and Orthotics Department [28]. Manchester Institute of Innovation Research [26] was consulted to consider responsible innovation and context transfer of the science of the project to expand beyond prosthetics and punctuated the importance of user-centred design. The British Association of Prosthetics and Orthotists (BAPO) [27] offered practical knowledge on the challenges faced by both clinicians and users, ensuring the project design aligns with real-world demands. The University of Salford’s Prosthetics and Orthotics Department [28] shared clinical expertise, explored prosthetic design and identified gaps in current prosthetic technologies. All these perspectives create a more user-centric design and appreciate the interconnectedness of scientific innovation with the practicalities of implementation and use of technology in society (See Human Practices).

2. Ideate

Utilising the insight from the aforementioned experts, a literature search was conducted to bridge the user problem of motion artefacts in prosthetics to a scientific solution [26-28]. Geobacter sulfurreducens, a Gram negative species of bacteria, form conductive e-pili that could reduce signal disruptions by providing seamless connection [29]. Furthermore, collagen binding proteins would provide a solution to electrodes slipping from their contact points on the skin [30]. Thus, reducing overall lagging limb movement by providing an uninterrupted connection between the muscle and prosthetic with consistent electrode contact with adhesion to skin.

3. Prototype

The aim was to create a proof of concept of skin-specific nanowires with collagen binding as a way to allow fluid prosthetic limb movement as the prototype. To create this, the wet lab process needed to be broken into different phases to address multiple considerations. The wet lab milestones are summarised below.

  1. Creation of an E. coli strain without fimbriae using CRISPR-Cas12a.
  2. Amplification of Type IV pili machinery genes from the E. coli K12 genome.
  3. Modification of the G. sulfurreducens’s pilA gene (gsPilA) by addition of protein-binding tag.
  4. Construction and introduction of plasmid with Type IV pili machinery and gspilA gene into E. coli NEB 5-alpha.
  5. Expression and test for e-pili formation.
  6. Harvesting of e-pili.

1. Creation of an E. coli strain without fimbriae using CRISPR-Cas12a.

The choice of chassis was E. coli due to its non-virulence as a commercial lab strain and its superior performance in cloning contexts, especially in DNA and protein yield. Specifically, we selected NEB5-alpha as our strain of choice due to its high transformation efficiency [31].

(See Safety to get deeper insight into how E. coli was chosen to be our chassis, and see Engineering to learn why we selected NEB5a as our final strain).

NEB 5-alpha has a fim operon that encodes its native type I pili [32] as well as multiple operons displaying homology with this fim operon. Out of these homologous operons, the sfm operon is most expressed in normal laboratory conditions [33]. As the focus is on obtaining only type IV pili from E. coli, it is necessary to prevent the production of native type I pili to increase yield of the desired pili. In these operons, the major structural subunit genes are fimA and sfmA respectively, and therefore, deleting these genes effectively prevents the formation of extracellular pili structures due to these operons. Thus, it is especially critical to procure a strain of E. coli NEB5a ΔfimAΔsfmA to serve as the chassis.

Unlike previous studies [35], we chose to employ a CRISPR-Cas12a system to delete other fimbriae-encoding genes due to the flexible Cas12a plasmid toolkit provided for gene deletion in E. coli by Jervis et al., 2021 [36].

2. Amplification of Type IV pili machinery genes from the E. coli K12 genome

After obtaining E. coli ΔsfmA to serve as the chassis and performing CRISPR to increase type IV pili yield, the type IV pili assembly machinery was needed. E. coli K12 genome was chosen to obtain the type IV pili machinery [36], and type IV pilus assembly genes were amplified from the E. coli K12 genome (Figure 6).

(More information on why the E. coli K12 genome was chosen is on the Safety page.)

Type IV pilus assembly gene fragements from k12
Figure 6. Type IV pilus assembly gene fragments from K12. The figure shows the identified and isolated four gene fragments of the type IV pili assembly machinery {see section what is IV pili to know more about the type IV pili machinery}. It was modified by the addition of Gibson assembly overhangs with Gibson assembly overhangs added for subsequent cloning steps in BglBricks pBbA1k and pBbE1c plasmids for co-transformation. Finally, it was amplified from the K12 genome using PCR.

3. Modification of gspilA gene by addition of protein-binding tag

Once the machinery was amplified from the K12 genome, the pili protein with addition of the protein binding tag for expression to create skin-specific nanowires was needed. So, the gspilA gene was modified to express the type IV by the addition of collagen binding protein tags. According to previous studies, the maximum number of residues that could be added at the C-terminus of the G. sulfurreducens pili monomer (GsPilA) while ensuring protein folding integrity and efficient protein assembly is retained in E. coli was 9 ≤ x < 14 [34,37]. Thus, this refined the choice of selection of binding tags, as it filtered the search to fit within this specific range. The collagen binding tags TKKTLRT and LRELHLNNN [21] were chosen as it fit this range. The modified E-pili with these collagen binding tags were expressed, as well as E-pili modified with a hexahistidine (His) tag for use as a control in protein detection.

4. Construction and introduction of plasmid with Type IV pili machinery and modified gspilA gene into E. coli NEB 5-alpha

There were two separate plasmids assembled using Gibson assembly to ensure protein expression and with the amplified Type IV machinery gene fragments sourced from the K12 genome and the modified gspilA (Figure 7). The genes in the first sub-plasmid included the ppdA-ppdB-ygdB-ppdC and gspO genes, while the second sub-plasmid contained the G. sulfurreducens’ pilA, hofB-hofC, and hofM-hofN-hofO-hofP-hofQ genes.

These plasmids were transformed into the prepared E. coli ΔsfmA.

Pili Expression Vectors
Figure 7. Pili expression vectors. The gene fragments ppdA-ppdB-ygdB-ppdC and gspO were assembled into a pBbA1k plasmid (A), while the fragments pilA, hofB-hofC, and hofM-hofN-hofO-hofP-hofQ were assembled into a pBbE1c plasmid (B). Created with Biorender.com

5. Expression and Test for E-pili presence

IPTG supplemented M9 media was utilised and plated the co-transformed E. coli ΔsfmA on IPTG supplemented M9 agar plates to induce protein expression [23]. This is because IPTG induces the lac promoter in the plasmid, thus expressing the desired protein [38]. Then, an SDS-PAGE was performed to test for all general protein expression for all modified pili, followed by a Western Blot for the His-tagged pili to isolate pili protein presence.

6. Harvesting of E-pili

The His-tagged E-pili was used as a diagnostic for protein presence by adapting it from Liu et al., 2020 [39]. The E-pili is purified by ammonium sulphate precipitation and an SDS page is performed to test for protein purity with the pili. If the His-tagged E-pili is sufficiently pure, then the same method to purify E-pili with other tags (collagen tags) is used. If the pili is not pure, an Ni-NTA affinity chromatography is performed on His-tagged E-pili to further purify (Figure 8).

Harvesting E-pili: through ammonium sulfate precipitation
Figure 8. Steps illustrating harvesting of E-pili. A) Protein nanowires are sheared from the cells in a blender. B) Cells are removed by centrifugation. The supernatant with the nanowires are precipitated by ammonium sulfate precipitation. C) Purity of pili verified using SDS Page. D) If pili are not pure, Ni-Nta Affinity Chromatography performed. Created with Biorender.com

4. Test

Now that the desired protein is expressed, it needs to be tested for its efficacy. Therefore a collagen binding assay and conductivity test for pili is needed to measure efficacy.

a) Test for binding to proteins

The collagen binding assay was adapted from Grenier et al., 1996 [40] to check for binding capability. This will be achieved by firstly transforming the pili-producing E. coli with a plasmid, pBbS1a, that codes for RFP. Then, the transformed cells are plated onto a collagen plate, washed, and the fluorescence reading is measured using a plate reader. In theory, E. coli which express the wild-type GsPilA should exhibit no fluorescence reading, while the E.coli that expresses the collagen-binding peptide will have a high fluorescence reading because the pili are interacting with the collagen and remain attached to the cell surface (Figure 9).

Illustration of our Collagen Binding Assay
Figure 9. Illustration of our Collagen Binding Assay. Cells transformed with RFP. High fluorescence wells depict binding of the cells expressing pili to collagen, while low fluorescence wells depict reduced pili-expressing cells binding to collagen. Created with Biorender.com

b) Test for conductivity

Due to the microscopic nature of these conductive E-pili, past literature [34, 41] designed custom devices for use in the conductivity measurement of E-pili proteins. The construction of these devices required many skills, notably electron-beam lithography and wire bonding, as well as an ample amount of time.

Our team lacked both the time and the expertise required to replicate these methods, so we seeked to design an alternative method to test for conductivity. With assistance from Prof. Thomas Thomson, an expert in nanoelectronics and spintronic technologies, we designed this simplified conductivity testing method (Figure 10) that was meant to be used to confirm whether the addition of a tag would impede the conductive nature of the E-pili protein [42].

Simplified conductivity set up
Figure 10. Simplified conductivity testing setup. Thin gold nanowires (A) will be placed as close together as possible on a microscope slide (B) through observation with a light microscope, and secured in place by varnish. A four-point probe measurement setup will then be created by linking the gold nanowires to a current source (C) and voltmeter (D), respectively. The purified E-pili solution (E) can then be drop-cast (F) onto the gold nanowires and conductivity can be measured by comparing the voltage across the electrodes from before and after the E-pili solution was drop-cast. Created with Biorender.com

5. Implementation

The implementation of the project after testing the collagen binding proof of concept is to use ceramides [43] as a binding tag on the surface of skin. This will be developed into a proper product that will be brought to market through the validation of clinical trials and testing [44].

Furthermore, biocompatibility tests are required to ensure the pili can integrate safely and effectively with biological tissues due to the product being classified as a prolonged exposure [45]. Thus it is imperative to consider factors when implementing the solution and giving the wider public access.

(Check out the Implementation Page to see our future outlook.)

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