Adding information to the parts registry

To help future iGEM teams that want to work with bioelectronic materials, we have added information to the registry parts from LINKS 2020 regarding the structure, assembly and potential applications of conductive pili. Even though our project is focused on Geobacter sulfurreducens pili, we have also researched other potential sources of conductive pili, such as Geobacter metallireducens and Pseudomonas aeruginosa. We have added the information we had gathered from our literature review to the parts BBa_K3552000, BBa_K3552001, and BBa_K3552002 in the sections labelled "Structure and function" and "Novel research and potential applications."

BBa_K3552000 (GsPilA)

Structure and function

The pili of Geobacter sulfurreducens are filaments primarily made up of the PilA protein monomer. Due to the short length of the monomer, aromatic residues (tryptophan, phenylalanine, and tyrosine) can be tightly packed along the pili’s length, providing it with conductive properties [1]. This characteristic can be explained by the phenomenon of pi stacking, in which the aromatic side chains align, creating a path for electron transfer to occur [2]. The relationship between the PilA's conductive properties and its protein sequence has further been proven by genetic engineering. For instance, conductivity of pili in which the five aromatic amino acids of the monomers are substituted with alanine is shown to drop to 38 ± 1 μS cm−1, three orders of magnitude lower than the native pili, underscoring the significance of the pi interactions in electron transfer [3]. Furthermore, pH also impacts the conductivity of pili; ranging from 51 mS/cm3 at pH 7 to 37 mS/cm3 at pH 10.5, with the highest conductivity value, 188 mS/cm3, observed at pH 2 [3]. Consistent with this, purified pili films demonstrate non-redox electronic conduction in a buffered aqueous environment; pili can conduct electrons without going through redox processes, which is in line with a metallic charge carrier transport model. This comes in sharp contrast to other organic reactions (e.g. DNA synthesis, photosynthesis), which follow a donor–bridge–acceptor model, where electric potential is passed on by the transfer of electrons from one molecule to the next until it reaches the final acceptor.This also explains how the conductivity of the pili is comparable to synthetic conductive materials, such as carbon nanotubes or silicon nanowires [4][5].

Novel research and potential applications

Over the last decade, research on pili has revealed a number of potential applications for G. sulfurreducens. For instance, microbial fuel cells can be constructed by connecting a G. sulfurreducens biofilm with the organic waste (e.g. acetate), generating electricity through pili-mediated reduction, [6]. Similarly, the capacity of natural Geobacter to reduce and detoxify radioactive elements and heavy metals in contaminated environments through pili-mediated reductions (e.g soluble Uranium VI to Uranium IV, a form that precipitates and is distributed at a lower rate) can also be enhanced, creating more efficient systems for clearing radiologically and chemically contaminated areas [7] [8]. Electrical biosensors, which could have medical applications in the future for the detection of relevant metabolic compounds, can be created by modifying the C-terminal end of the pilin monomers. Hybrid pili, which consist of wild type, as well as his-tagged and HA-tagged monomers have been shown to conduct electricity better than the wild type pili, as well as change their conductivity potential when binding to the target molecule nickel [9] [10]. Finally, G. sulfurreducens can serve as a model organism for microbially-induced corrosion in metal structures, such as storage tanks and pipes; understanding the electrochemical mechanism of this process can pave the way for technologies that lower maintenance requirements and increase the longevity of these buildings [11].

BBa_K3552001 (GmPilA)

Structure and function

PilA protein subunits make the pili of the anaerobic species Geobacter metallireducens . Because of the dense arrangement of aromatic amino acids, which promote electron transport over the length of the pilus through the phenomenon of pi-pi stacking, these protein filaments are some of the most conductive naturally occurring biological materials known [12]. These conductive pili support G. metallireducens metabolism by providing an electron transfer link between iron oxides, which are reduced, and various organic compounds, such as toluene, acetate and ethanol, which are oxidised [13].

Novel research and potential applications

Recent studies have shown that Geobacter metallireducens pili have conductivities that are noticeably higher than those of other Geobacter species. For example, heterologous expression of the pilA gene from G. metallireducens in Geobacter sulfurreducens resulted in pili that exhibited conductivities that were 5000 times higher than those of Geobacter sulfurreducens, the most characterised member of the family [12]. The improved conductivity can be explained by the higher percentage of aromatic amino acid residues in the Geobacter metallireducens pilin (15.3% compared to 9.85% in G. metallireducens) [14]. Similarly to G. sulfurreducens, G. metallireducens pili's conductive qualities may be useful in a variety of contexts, including bioelectronic sensors and environmental cleanup devices [15]. Although most modern research has focused on G. sulfurreducens and its capacity to detoxify radioactive elements and metabolise pollutants, Geobacter metallireducens’s naturally higher conductivity may prove to be a more useful candidate for future biotechnological applications.

BBa_K3552002 (PaPilA)

Structure and function

Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium and one of the most common causes of hospital-acquired infections [16]. As an opportunistic pathogen, it utilises its pili and flagella for biofilm formation and cell-surface adhesion [17]. The assembly of the monomeric subunit PilA, which is localised on the surface of the cell and results in type 4 pili [18].

Novel research and potential applications

P. aeruginosa pili, similarly to those of the Geobacter genus, have the potential to be modified for use in bioelectronics. The N-terminal alpha-helix domain of type IV pili monomers is highly conserved across multiple bacterial species and is shown to have very similar conductive properties to G. sulfurreducens pili [19]. According to the same research, substituting glutamate, leucine and glycine with tyrosine and phenylalanine residues has shown to significantly increase the pili’s conductivity, highlighting their importance in the electrical activity of the pili. Even though P. aeruginosa is not an ideal candidate for large-scale pili production due to its pathogenicity, the natural adherence of these pili to different biological surfaces could potentially be used to manufacture biocompatible electronics in the future if expressed in a non-pathogenic chassis, such as E. coli [20].

Designing plasmids for chromosomal deletion of sfmA and fimA in E. coli

Another important aspect of our project was utilising a CRISPR-Cas12a system to delete both the sfmA and fimA gene from the E. coli NEB5a genome. As such, we have added the fragment cassettes of the pTF_sfmA and proposed pTF_fimA plasmids that contain the gene-specific spacer DNA and homology arms as parts for future teams to use. With pTF_sfmA, we have achieved a sfmA gene deletion efficiency of 50%; the pTF_fimA plasmid is untested but is designed following the same method as the pTF_sfmA plasmid. The CRISPR system we have used [21] is highly adaptable to different protocols, so we hope that we can inspire other iGEM teams to use the system for their own gene deletion experiments.

Part	Description
BBa_K5177024	pTF_sfmA plasmid fragment cassette
BBa_K5177025	Proposed pTF_fimA plasmid fragment cassette