Proof of Concept

Intro

Proof of concept shows that the engineered system or construct can achieve the desired function or outcome.

  • Summarisation of key experiments that validate the goal of the project
  • Evidence that the system performs as intended, including any quantitative or qualitative results
  • Examples where the design successfully meets a specific need or challenge
  • Discussion of the broader implications of the successful proof of concept for the project’s goals

Key experiments

We planned a series of experiments to validate the system, specifically:

  • Sequencing of plasmids to confirm correct assembly
  • Western blot to confirm the expression of pili
  • Collagen-binding assay to test the functionality of the engineered pili containing the collagen-binding peptides
  • Conductivity testing to assess the impact of adding a tag on the pili’s conductivity
However, due to time constraints, only the assembly of the vectors could be confirmed and the collagen-binding assay was conducted. While the sequencing was successful, the collagen-binding assay did not yield the expected results.

Construction of vectors

The gene encoding the main protein monomer of the pili, derived from Geobacter sulfurreducens (gsPilA) , was amplified by PCR and modified to include either a sequence for a His-tag or collagen-binding motif (TKKTLRT or LRELHLNNN). Additionally, four gene fragments, collectively encoding the type IV pilus assembly system, were amplified by PCR from Escherichia coli K12 (MG1655). These fragments were then assembled into two distinct plasmids (Figure 1).

Figure 1 Pili Expression Vectors
Figure 1. Pili expression vectors.[A] The gene fragments ppdA-ppdB-ygdB-ppdC and gspO were assembled into a pBbA1k-based vector.[B] The fragments gsPilA, hofB-hofC , and hofM-hofN-hofO-hofP-hofQ were assembled into a pBbE1c-based vector. Created with Biorender.com

All components were successfully cloned into their respective vectors for co-transformation.

Collagen Binding Assay

A collagen-binding assay was conducted as described in the Results page. However, the difference in fluorescence between collagen-coated and non-coated plates was not statistically significant for any of the strains.

Test for E-pili Conductivity

We also sought to test for the conductivity of our e-pili. Previous studies [1, 2] have created measurement devices composed of nano-scale gold electrodes linked to a voltage source in order to measure the conductivity of individual e-pili proteins. However, we realized after discussion with Prof. Thomas Thomson, a Professor of Nanoelectronics at our University, that the techniques involved with creating such a measurement device, notably electron-beam lithography and wire bonding, required years of practice to master, and even with complete mastery, needed weeks of time to perform. The sheer scale difference between our microscopic e-pili and most of the accessible electrical measurement devices posed a significant challenge for our development of a feasible method that matched our level of expertise. Thankfully, with assistance from Prof. Thomson, we were able to design this simplified conductivity testing method which might be sufficient to demonstrate whether the addition of a binding tag would impact the overall conductivity of our pili. Figure 2 is representative of our overall conductivity testing method.

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Figure 2.Simplified conductivity testing setup. Thin gold nanowires [A] will be placed as close together as possible on a microscope slide [B] 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 from after the e-pili solution was drop-cast. Created with Biorender.com

Because of insufficient cell growth that impeded the progression of our experiments, we did not have enough time to construct this system and perform actual measurements. However, this method may prove useful to future iGEM teams who wish to test for protein conductivity but do not have the required skills and resources to follow the methods from previous studies.

Broader Implications

Although the pBbA1k and pBbE1c vectors were successfully constructed and co-transformed into E. coli , a Western Blot was not conducted to confirm the expression of the engineered pili. Challenges encountered in the collagen-binding assay may stem from issues with pili production. The absence of clear binding results suggests that the pili might not have been expressed as expected (see Engineering) . This may highlight the need for further optimisation of the expression system, which may be crucial for enhancing the reliability of pili production. To address this, future iterations of the project could focus on refining vector construction with different promoters or adjusting culture conditions. Additionally, exploring alternative binding peptides or materials could improve the specificity and strength of binding, enhancing the overall performance of the nanowires (see Results). The development of e-pili for improved myoelectric prosthetics could significantly enhance the functionality and usability of prosthetic devices. By reducing motion artifacts and improving signal fidelity, users may experience more intuitive and responsive limb control. With more reliable control over prosthetic limbs, patients could also engage in physical therapy more effectively, leading to faster recovery times and improved adaptation to their devices. Beyond its immediate application to prosthetics, the principles behind E.lectrode could extend to other medical fields that utilise electromyography (EMG) signals, such as neurological rehabilitation, wearable health monitoring devices, and even brain-computer interfaces (see Implementation).

References