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Item type:Publication, Access status: with files , Living electronics: coupling S. oneidensis MtrC-SpyTag cells to SpyCatcher-functionalized electrodes for direct electron transfer(Elsevier, 2026-08-15); ; ; ; This work set out to establish an easily applicable system to improve a broad range of bioelectronic devices using the SpyTag-SpyCatcher crosslinking system together with one of the model organisms for extracellular electron transport, Shewanella oneidensis . Therefore, the surface-displayed c -type cytochrome MtrC was equipped with an accessible SpyTag and coupled to SpyCatcher-functionalized surfaces. A transposon screen followed by nanopore sequencing was conducted in order to identify integration positions which facilitate MtrC functionality while the SpyTag is surface accessible. Three integration positions (W314, Y417, T603) were chosen for further characterization. Expression of the MtrC-SpyTag constructs in a S. oneidensis strain lacking all outer membrane cytochromes restored the ability to reduce an extracellular electron acceptor. Two of the three strains reached reduction rates at the wildtype MtrC level proving that the integrated SpyTag does not hamper extracellular electron transfer. In vivo, all three constructs showed significantly better binding properties to SpyCatcher functionalized magnetic beads than the wildtype MtrC control. The two most promising candidates were coupled to conductive, magnetic gold nanoparticles and directed towards a screen-printed electrode showcasing how MtrC-SpyTag expression can improve bioelectronic devices. A significantly higher charge transfer compared to the wildtype control was reached in linear sweep voltammetry and chronoamperometry experiments. Moreover, a shift towards direct electron transfer was observed which reduces the problem of redox shuttle washout in flowthrough systems. Direct binding of the cells to SpyCatcher functionalized electrodes enabled robust current production even after thorough washing of the electrodes while control cells failed to produce current under these conditions. The versatile SpyCatcher toolbox can be used together with the here reported strains to eliminate bottlenecks in bioelectronics like poor biofilm formation or the production of an insulating extracellular polymer matrix.Publicationtype: Journal Article47 14 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexesShewanella oneidensis is known for its ability to respire on extracellular electron acceptors. The spectrum of these acceptors also includes anode surfaces. Based on this activity, a versatile S. oneidensis based biosensor strain was constructed in which electricity production can be modulated. Construction started with the identification of a usable rate-limiting step of electron transfer to an anode. Thereafter, the sensor strain was genetically engineered to produce a protein complex consisting of the three proteins MtrA, MtrB and MtrF. This complex is associated to the outer membrane and most probably enables membrane spanning electron transfer. MtrF is an outer membrane cytochrome that catalyzes electron transfer reactions on the cell surface. Under anoxic conditions, wild type cells do not express MtrF but rather MtrC as electron transferring outer membrane cytochrome. Still, our analysis revealed that MtrF compared to MtrC overexpression is less toxic to the cell which gives MtrF a superior position for biosensor based applications. Transcription of mtrA, mtrB and mtrF was linked up to an inducible promoter system, which positively reacts to rising l-arabinose concentrations. Anode reduction mediated by this strain was linearly dependent on the arabinose content of the medium. This linear dependency was detectable over a wide range of arabinose concentrations. The l-arabinose biosensor presented in this study proves the principle of an outer membrane complex based sensing method which could be easily modified to different specificities by a simple change of the regulatory elements.Publicationtype: Journal ArticleScopus© Citations 79 12 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , A versatile miniature bioreactor and its application to bioelectrochemistry studies(Elsevier, 2010-04-21); ; ; ; Often, reproducible investigations on bio-microsystems essentially require a flexible but well-defined experimental setup, which in its features corresponds to a bioreactor. We therefore developed a miniature bioreactor with a volume in the range of a few millilitre that is assembled by alternate stacking of individual polycarbonate elements and silicone gaskets. All the necessary supply pipes are incorporated as bore holes or cavities within the individual elements. Their combination allows for a bioreactor assembly that is easily adaptable in size and functionality to experimental demands. It allows for controlling oxygen transfer as well as the monitoring of dissolved oxygen concentration and pH-value. The system provides access for media exchange or sterile sampling. A mass transfer coefficient for oxygen (kLa) of 4.3×10-3s-1 at a flow rate of only 15mlmin-1 and a mixing time of 1.5s at a flow rate of 11mlmin-1 were observed for the modular bioreactor. Single reactor chambers can be interconnected via ion-conductive membranes to form a two-chamber test setup for investigations on electrochemical systems such as fuel cells or sensors. The versatile applicability of this modular and flexible bioreactor was demonstrated by recording a growth curve of Escherichia coli (including monitoring of pH and oxygen) saturation, and also as by two bioelectrochemical experiments. In the first electrochemical experiment the use of the bioreactor enabled a direct comparison of electrode materials for a laccase-catalyzed oxygen reduction electrode. In a second experiment, the bioreactor was utilized to characterize the influence of outer membrane cytochromes on the performance of Shewanella oneidensis in a microbial fuel cell.Publicationtype: Journal ArticleScopus© Citations 20 15