Biomimetic engineering of conductive curli protein films

Abstract

Bioelectronic systems derived from peptides and proteins are of particular interest for fabricating novel flexible, biocompatible and bioactive devices. These synthetic or recombinant systems designed for mediating electron transport often mimic the proteinaceous appendages of naturally occurring electroactive bacteria. Drawing inspiration from such conductive proteins with a high content of aromatic residues, we have engineered a fibrous protein scaffold, curli fibers produced by Escherichia coli bacteria, to enable long-range electron transport. We report the genetic engineering and characterization of curli fibers containing aromatic residues of different nature, with defined spatial positioning, and with varying content on single self-assembling CsgA curli subunits. Our results demonstrate the impressive versatility of the CsgA protein for genetically engineering protein-based materials with new functions. Through a scalable purification process, we show that macroscopic gels and films can be produced, with engineered thin films exhibiting a greater conductivity compared with wild-type curli films. We anticipate that this engineered conductive scaffold, and our approach that combines computational modeling, protein engineering, and biosynthetic manufacture will contribute to the improvement of a range of useful bio-hybrid technologies.

Challenges in engineering conductive protein fibres: Disentangling the knowledge

“…[18,[36][37][38][39][40] Dissimilar experimental conditions such as the state of the material and surrounding environment have been highlighted as possible culprits. [29,30,41,42] The recent expansion of the field into the engineering of novel conductive protein materials has been based on design principles directly inspired by G. sulfurreducens, [19][20][21][22] making it all the more critical that G. sulfurreducens' electronic behaviour is well-understood. Without understanding how experimental conditions impact measurements, the rational design, accurate comparison, and evaluation of novel conductive protein materials become infinitely more difficult.…”

“…[37] This rationale has been proven effective in engineering curli fibres where proteins with denser aromatic amino acid residues profiles were found to be more conductive. [19] Protein folding also decreases the reorganization energy needed in redox reactions in cytochrome electron transfer. [84] Just like the proximity of aromatic rings increases conductivity, cytochrome groups can be similarly affected.…”

“…[22] Modified curli fibre proteins from E. coli with an attached aromatic rich peptide sequence displayed linear responses ( Figure 5C), [21] while aromatic substitutions on the protein itself displayed a transition from linear to power law depending on the voltage applied ( Figure 5D). [19] Small differences in measurement setup may explain some of the differences in result. The voltage range used varied from ±0.8 to ±5 V. The dependence of how a specific conduction mechanism responds to an applied potential as previously described may account for these differences in result.…”

“…In fact, various types of synthetic conductive protein fibres have recently been engineered using recombinant DNA technologies and engineered microbes as chassis. [19][20][21][22] These novel conductive biomaterials hold Sophia Roy and Oliver Xie contributed equally.…”

Living materials with programmable functionalities grown from engineered microbial co-cultures

“…The nascent field of engineered living materials (ELMs) aims to recapitulate the desirable properties of natural living biomaterials to create novel, useful materials using geneticallyengineered organisms [1][2][3][4] . Most ELMs to date have either synthesised novel materials from natural macromolecules and polymers harvested from microbial cells [5][6][7][8][9][10][11][12][13][14] or have made use of the multiple functionalities of living cells by embedding these within man-made hydrogels [15][16][17][18][19][20] . However, the long-term goal of ELMs research is to use engineered cells, rationally reprogrammed at the DNA level, to both make the material and incorporate novel functionalities into it at the same time -thus 'growing' functional biomaterials in situ 3 .…”