Engineering E. coli to enable electron transfer along curli protein nanofibers
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1
Harvard University, Wyss Institute for Biologically Inspired Engineering, United States
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2
Harvard University, School of Engineering and Applied Sciences, United States
Protein-based materials capable of mediating long-range electron transport are attractive for incorporation in several devices including biosensing, light harvesting, and electrobiosynthetic systems, as well as microbial fuel cells and other electronic devices. These technologies can benefit from the flexibility, bio-compatibility, nano-size, and self-assembling properties of protein materials. Some conductive extracellular appendages are naturally produced by microorganisms like Shewanella and Geobacter, but it is difficult to improve upon their intrinsic conductivity via rational design because of the difficulty to genetically engineer these organisms, the limited variety of functional groups of natural amino acids, and the lack of structural information available. Here, we propose to use Escherichia coli as a more versatile platform for the production of conductive protein fibers. E. coli can be easily engineered, is routinely grown under aerobic conditions, and naturally produces curli fibers composed on repeated CsgA proteins. CsgA has an amyloid structure and spontaneously assembles into microns-long nanofibers. Engineering the CsgA gene allows for the addition of various active protein domains along the fibers, or for the selective mutation of residues that change surface chemistry. The curli nanofibers can thus be used as a scaffold for the creation of functional porous fibrous networks.
In order to generate electron transfer, we aimed at creating pi-pi stacking along curli fibers. We identified residues in CsgA that can be mutated without altering its amyloid structure, and then selected series of residues to mutate aligned along the five repeats of the beta-helix structure of CsgA. We postulated that the conjugated side-chains of aligned aromatic residues, or their non-natural amino acid derivatives, could interact, form pi-stacks, and induce electron delocalization. Congo red binding assays and electron microscopy indicate that mutant CsgA proteins with various sets of aligned aromatic residues can still form amyloid curli fibers. The engineered bacteria can be used directly to grow biofilms, or alternatively His-tagged curli fibers are purified to investigate the properties of single nanofibers. The ability of the mutant fibers to transfer electrons is investigated via conductive AFM and electrochemical methods, comparing with naturally-occurring conductive nanowires.
Overall, our engineered E. coli system provides a tunable platform for the synthesis of conductive protein networks. Diverse functional peptides could be further introduced in the curli scaffold, such as metalloproteins, redox enzymes, binding domains, and stimuli-responsive peptides, to add additional functionalities. This versatile biomaterial could be used in a variety of devices both for biomedical or energy applications.
Keywords:
Biomimetic,
nanofiber,
electric,
engineered cell
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
Poster
Topic:
Nano-structured materials for unique functions
Citation:
Dorval Courchesne
N,
Tay
PR,
Nguyen
PQ and
Joshi
NS
(2016). Engineering E. coli to enable electron transfer along curli protein nanofibers.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.01972
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.