Engineering protein components for living electronics

Living cells sense and respond to an astounding array of different molecules. Integrating these cells into digital devices to produce living electronics has the potential to create useful devices, such as bioelectronic sensors that combine the sensitivity and specificity of biological systems with the capabilities of conventional electronics. While there are diverse standardized silicon semiconductor components for electrical engineering, biological systems lack equivalents, which limits the functions of current bioelectronic devices. Protein electron carriers are attractive targets to adapt as bioelectronic components because they are mutable and because their output, electron transfer, is easy to interface with electronics. Herein, I describe my efforts to engineer ferredoxin and flavodoxin electron transfer proteins as components in controlling electron flow in cells. In these studies, I probe the tolerance of a cyanobacterial flavodoxin to insertion of a small octapeptide, which elongates the primary structure. I find that flavodoxin sites do not tolerate insertion if they are proximal to residues that mediate cofactor or protein partner interactions. Additionally, I use protein engineering to create an allosteric ferredoxin through insertion of an anti-GFP nanobody. I demonstrate that a ferredoxin which contains a specific anti-GFP nanobody insert requires co-expression with GFP to display electron transfer activity. These studies lay the groundwork for further development of biological components for living electronics. Mutation-tolerant sites identified in the flavodoxin study may be targeted for further engineering to produce allosteric flavodoxins. Additionally, the GFP-dependent ferredoxin produced demonstrates that insertion of nanobodies is a viable strategy for controlling protein activity through specific protein-protein interactions. Further successful application of this strategy has the potential to greatly expand our ability to regulate protein electron transfer for bioelectronics applications.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Natural Sciences

Grantor

Rice University

Year dc:date.issued

2024

Author dc:creator

• Truong, Albert

Advisors dc:contributor.advisor

• Silberg, Jonathan J.
• Ajo-Franklin, Caroline

• flavodoxin
• ferredoxin
• nanobody
• protein engineering
• cellular selection
• bioelectronics
• peptide insertion
• laboratory evolution
• allostery

Statement dc:rights

• Copyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1911/118372

OAI identifier oai:identifier

oai:repository.rice.edu:1911/118372