---

Overview

We added new information learned from the literature and our experimental results to the previous parts listed below:

arsR (BBa_J15101)

A relatively well-studied arsenic resistance operon is the one found in Escherichia coli, which contains arsR (transcriptional regulator), arsB (arsenite permease), and arsC (arsenate reductase)^[1]. When arsenic is absent, the transcription regulator ArsR binds to the ArsR-binding site (ABS) within the ars promoter and blocks transcription. Once arsenic is present, it binds to ArsR and activate the transcription of the ars genes and clear arsenic in the cell^[2]. The arsR regulator and the promoter of this operon have been used to construct arsenic whole cell biosensors (WCB) in various microorganism hosts.

When arsenic is absent, the transcription regulator ArsR binds to the ArsR-binding site (ABS) within the ars promoter and blocks transcription. Once arsenic is present, it binds to ArsR and activate the transcription of the downstream genes^[3,4]. We used gfp as a reporter to test its function of arsenic detection. Since this ars system comes from E. coli genome, to eliminate this impact, we used another model bacteria Shewanella oneidensis as the chassis cells to verify the function of this part. As the result shows below, with the arsenic concentration rises, the strain with the reporter produced higher fluorescence intensity.

Fig. 1 Fluorescence curve of the reporter strain with different arsenic concentrations

mtrC (BBa_K3102020)

We found some interesting information from the articles that explain the function of MtrC in the process of S. oneidensis extracellular electron transfer. S. oneidensis MR-1 was among the first identified microorganisms capable of using minerals that contain Fe(Ⅲ), Mn(Ⅲ) or Mn(Ⅳ) as terminal electron acceptors. Genetic studies of this bacterium revealed the direct involvement of six multihaem c-Cyts CymA, Fcc3 (also known as FccA), MtrA, MtrC, OmcA and small tetrahaem cytochrome (STC) — and the porin-like outer membrane protein MtrB in the extracellular reduction of minerals that contain Fe(Ⅲ). Functional characterization has confirmed that CymA oxidizes quinol in the cytoplasmic membrane and transfers the released electrons to the periplasmic c-Cyts Fcc3 and STC^[5]. Because a mutant without Fcc3 and STC has an impaired ability to reduce Fe(Ⅲ) oxides or oxyhydroxides, both Fcc3 and STC are proposed to transport electrons from CymA to MtrA. MtrA, MtrB and MtrC form a trans-outer membrane protein complex that transfers electrons from the periplasmic proteins to the bacterial surface. Finally, on the bacterial surface, MtrC and OmcA can physically interact with each other and transfer electrons directly to minerals that contain Fe(Ⅲ), probably through solvent-exposed haems. Notably, MtrC and OmcA also associate with extracellular structures that were previously referred to as ‘nanowires’ . Recent results have demonstrated that nanowires are extensions of the outer membrane that contain MtrC and OmcA and which can make physical connections with neighbouring cells. These outer membrane extensions are proposed to mediate the transfer of electrons to minerals and other S. oneidensis MR-1 cells through a multistep hopping mechanism^[6].

Fig. 2 Metal reducing pathways (Mtr) of S. oneidensis^[5]

cymA (BBa_K2706008)

Microbial electron exchange with extracellular substrates, such as electrodes in BESs, is often called microbial extracellular electron transfer (EET). The most well characterized microbial EET pathway is the Mtr pathway of S. oneidensis MR-1. S. oneidensis MR-1 transfers electrons from the quinone/quinol pool in the cytoplasmic membrane to the bacterial surface through redox and structural proteins, including the inner membrane c-Cyt CymA, the periplasmicc-Cyts Fcc3 and STC, and the outer membrane porin-c-Cytcomplex MtrCAB^[5]. Eventually, electrons are transferred from the bacterial surface to extracellular acceptors directly via the outer membrane c-Cyts or via nanowires, or indirectly via self-secreted electron mediator flavins. Shewanella nanowires are the extensions of the outer membrane and periplasm in which the cell surface c-Cyts MtrC and OmcA are the key players for long-distance electron transfer^[5,6].

Fig. 3 Extracellular electron transfer (EET) pathways of S. oneidensis MR-1 to electrodes^[6].