Abstract

Bioelectrochemical sensors, in which electrochemically active biofilms (EABs) are employed as bio-elements, sense analytes of interest by converting metabolic changes to easily detectable electrical signals. EABs are formed by electroactive bacteria capable of exchanging electrons with electrodes. The self-immobilization, self-sustainability, and high robustness of EABs make EAB-enabled biosensors show promise in environmental applications, such as water quality monitoring. This year, our team utilized the electroactive microorganism Shewanella oneidensis MR-1 as the chassis cell to construct an EAB-based biosensor for sensitive and specific detection of arsenic.

Firstly, we engineered the biofilm of S. oneidensis MR-1 by knocking out its ptpA gene that is associated with the biosynthesis of extracellular polysaccharides, resulting in a more conductive S. oneidensis MR-1 biofilm formed on the anode of a microbial fuel cells (MFCs). The enhanced conductivity of S. oneidensis MR-1 biofilm improved the sensitivity of EAB-based biosensor for arsenic detection. However, the biosensor exhibited a low specificity for arsenic detection.

To improve the specificity of our biosensor, we selected the P_ars-arsR system from Escherichia coli as the arsenic sensing components. This arsenic responsive transcription system was used to control the expression of cytochrome genes cymA or mtrC that is involved in extracellular electron transfer in S. oneidensis MR-1. By transferring this gene circuit into double deletion strains ΔptpAmtrC or ΔptpAcymA, the presence of arsenic can be converted to increased electrical signals, as arsenic can promote the expression of cymA or mtrC to recover the ability of EET in S. oneidensis MR-1.

To further optimize the biosensor, we construct a self-amplifier system that does not rely on exogenous inducers (see Part improve for details) by modifing the LuxR protein from Vibrio fischeri. We combined this self-amplifier system with the arsenic sensing circuit to enhance the sensitivity and specificity of the biosensor.

Taken together, this work includes three phases: engineering of S. oneidensis electroactive biofilm, construction and validation of the arsenic sensing system, and construction of the self-amplifier system.

Phase 1: Engineering of S. oneidensis Electroactive Biofilm

The electroactive biofilm is composed of electroactive cells and the extracellular polymeric substances (EPS) they secrete. EPS mainly consists of extracellular polysaccharides, proteins, DNA, and electroactive compounds such as cytochromes and nanowires. Among these components, extracellular polysaccharides is unconductive, resulting in inefficient EET. The ptpA gene plays a key role in the synthesis of extracellular polysaccharides. Thus, the deletion of ptpA can reduce the amount of extracellular polysaccharides in biofilm matrix to enhance the conductivity of the S. oneidensis MR-1 biofilm. It has been reported that the enhanced EET efficiency of EAB can improve the sensitivity of EAB-based biosensors. Thus, we knocked out the ptpA gene from the S. oneidensis MR-1 genome and applied ΔptpA in MFCs. The results showed that ΔptpA produced higher electrical generation than the WT, and ΔptpA biofilm exhibited higher conductivity. To test if this conductive biofilm can be used as sensitive elements to improve the sensitivity of sensor, we comparatively analyzed concentration-response curves of MFCs inoculated with S. oneidensis MR-1 wild type and ΔptpA via As³⁺ shocks. The results show that the EAB-based sensors with ΔptpA biofilms can detect lower concentrations of As³⁺ than ones with wild type biofilms.

However, other heavy metal, such as cadmium, also can change electrical signal of sensor, indicating low specificity of this S. oneidensis biofilm-based biosensor.

Fig 1. ptpA gene knockout verification

Phase 2: Construction and Validation of the Arsenic Sensing System

To improve the specificity of sensor, we cloned an arsenic responsive transcription system, i.e., the P_ars-arsR system to control the expression of EET-associated genes. The arsenic-inducible promoter (P_ars) is negatively regulated by ArsR. When arsenic is excluded from a cell with these genetic components, the binding kinetics between ArsR and its associated operator region within P_ars are relatively strong, blocking the transcription of downstream genes. The dynamics of the circuit change, however, when arsenic enters the cell. In this condition, arsenic associates with ArsR, inducing a conformational change that leads to its dissociation from the P_ars operator. Because the binding kinetics between ArsR and the ABS become more unfavorable with increasing arsenic concentrations, GFP levels increase with increasing arsenic presence. To verify that the P_ars-arsR system from E.coli can work properly in Shewanella cells, we firstly fused arsR with gfp to obtain Part 1 (Fig.2). Theoretically, when arsenic concentration increases in the system, fluorescence intensity increases (see results).

Fig 2. Structure of part 1 and Colony RPC result of part 1

The cymA and mtrC genes encode c-type cytochromes that play important roles in EET of S. oneidensis MR-1. Previous studies showed that the deletions of cymA or mtrC disrupted the ability of S. oneidensis MR-1 to generate electricity in MFCs. We knock out the cymA and mtrC genes from ΔptpA strain, obtaining the double knockout strain ΔptpAmtrC or ΔptpAcymA. Meanwhile, we fused cymA and mtrC with the P_ars-arsR system, respectively, obtaining Part 3 and Part 4 (Fig.3). We transferred Part 3 plasmid and Part 4 plasmid into ΔptpAmtrC or ΔptpAcymA. By conducting MFCs experiments, we found that when the cultures of ΔptpAcymA with Part 3 and ΔptpAmtrC with Part 4 were induced by arsenic with different concentrations, the electricity signals enhanced with the increase in arsenic concentrations. However, the cadmium cannot change electricity signals. It indicates that this biosensor can detect specifically detect arsenic (see the result).

Fig 3. Structure of part 3/4 and Colony RPC result of part 3/4

Phase 3: Building Amplifiers to Boost Biosensor Performance

To further improve the sensitivity of our biosensor, we constructed the P_luxI-luxR (Δ2-162) positive feedback system (See Project Design) and combined it with the arsenic-responsive transcription system, which were conducted in the cycle 2. Firstly, we fused luxR (Δ2-162) with gfp to get Part2 (Fig. 4) and compared it with the original luxR. It was verified that it could drive downstream gene expression without the addition of the exogenous AHL in S. oneidensis MR-1 (See improved parts).

Fig 4. Structure of part 2 and Colony RPC result of part 2

Then, we constructed Part5/6/7 and fused cymA and mtrC with luxR (Δ2-162) to obtain the corresponding gene circuis Part 6,7 (Fig5). In the part 5, we used luxR (Δ2-162) as the reporter gene in response to the arsenic. With the arsenic level rises, part 5 will produce more LuxR (Δ2-162) as a result. Then the LuxR (Δ2-162) functions as an activator to start the positive feedback loops of part 6/7. LuxR (Δ2-162) can active the gene transcription driven by the lux promoter in part 6/7, producing MtrC/CymA and more LuxR (Δ2-162). In this case, LuxR (Δ2-162) works as both reporter and activator, creating a positive feedback loop gene circuit.

Fig 5. Structure of part 5,6,7 and Colony RPC result of part 5,6,7

Finally, we insert Part 6/7 into the back of Part 5 to obtain the final cymA-enchanced biosensor Part 8 and mtrC-enhanced biosensor Part 9 (Fig 6). After the modification of the luxR (Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, luxR (Δ2-162), was used to replace mtrC as reporter, and it was regulated by the arsR-P_ars circuit, while mtrC/cymA together with a second luxR (Δ2-162) was placed under the promoter P_luxI, which was activated by luxR (Δ2-162). When arsenic is present, it activates the expression of luxR (Δ2-162) in the first circuit, which turns on the expression of mtrC/cymA and luxR (Δ2-162) (Part 5)from the following circuit. The second luxR (Δ2-162) activates its own expression as well as that of mtrC/cymA and forms a positive feedback loop to enhance the output signal from mtrC/cymA. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier (Part 6 & 7). Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates and better performance in MFC, increasing the detection range, and improving sensitivity.

Fig 6. Structure of part 8/9 and Colony RPC result of part 8/9