Part:BBa_K957000

Arsenic inducible mtrB with cut sites flanking RBS

The purpose of this part is to upregulate mtrB expression in response to arsenic. When expressed in a Shewanella strain lacking mtrB on the chromosome, this composite part will function as a component of a biosensor. Specifically, when such a complemented strain is inoculated in a microbial electrochemical system, current output will increase in response to arsenic.

Within the genetic circuit, arsR acts as a negative autoregulator, repressing not only the expression of downstream mtrB, but also its own expression. Upon association with arsenic salts, ArsR dissociates from the operator of the arsenic inducible promoter, upregulating expression of downstream protein. Because mtrB activity is requisite for functionality of the mtr electron pathway in Shewanella spp., arsenic thus induces electron shuttling through the mtr pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 255
  Illegal BamHI site found at 491
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Overview of Characterization in Bioelectrochemical Systems

As described on the 2012 Cornell iGEM wiki section, S. oneidensis MR-1 is capable of shuttling electrons through the Mtr pathway to reduce extracellular metals because of the negative free energy change associated with these redox reactions. Thus, to encourage Shewanella to transfer electrons to an electrode, we poise the potential of an electrode in a three electrode system, controlled by a potentiostat, so that electron transfer is energetically favorable to the organism. In short, a potentiostat works by setting the potential of a working electrode (WE) with respect to a Ag/AgCl reference electrode (RE) by injecting current through a counter electrode (CE).

Because we are interested in continuous monitoring of contaminants, characterization focused on the operation of reactors in continuous flow setup, wherein reactors approached steady state current outputs at each level of analyte. In general, all experiments were set up in bench-scale reactors provided by the Angenent Lab, with a constant fluid volume of 120mL and a consistent electrode-surface area. All characterization experiments began at an analyte concentration of zero, as media was fed to the system at a constant rate of 18 mL/min. Once a system reached steady state—for a period of greater than three system retention times—the analyte concentration in the feed tank was increased. By repeating this process after new steady state current outputs were reached, we were able to characterize the current response of our reporter and control strains to either arsenic-containing compounds or naphthalene, as appropriate.

After initial characterization of our arsenic-sensing strains, we have shown that our arsenic sensor works as expected, producing higher levels of steady-state current in response to arsenite salts. However, more trials are needed in order to construct a reliable calibration curve.

Arsenic Sensing

We chose to initially characterize our arsenic-sensing Shewanella by dosing with sodium arsenite, since the organism's native arsenate reductase activity would introduce a confounding variable in part characterization. After this initial characterization , we have shown that current is, indeed, upregulated in response to our analyte of interest. However, we should emphasize that this data is preliminary: Because of the care we took to establish a thorough Standard Operation Procedure for the handling of arsenic, we only had time for a single characterization trial, shown below in Fig. 4. Additionally, we plan on characterizing in response to both arsenate salts (to determine whether arsenate reductase activity is indeed confounding) and antimonite (to estimate the likelyhood of false positives).

Fig. 4. Current production over time is plotted for continuous flow reactors inoculated with our arsenic-sensing strain (JG700+p14k). Transient phases corresponding to arsenite concentrations of 100 μM (blue) and 500 μM (green)are shown. Before a potentiostat channel restart at time zero, basal current was recorded at approximately 4 μA. We report induced current in percent of this basal response.

It is also important to point out that we have not normalized any data to a per-cell basis. Because we are interested in a field-deployable system that produces greater absolute current in response to analyte, we did not record either optical density or colony forming units over time. However, while not a rigorous, we have observed a visible decrease in biomass for increasing concentrations of arsenite—as would be expected. Therefore, it is likely that an a per-cell basis, our arsenic sensing strains produce much more current that Fig. 4 would suggest. We plan on repeating characterization experiments in response to arsenite—both to generate a reliable transfer function relating current to arsenite concentration and to better understand the relationship between specific growth rate and MtrB production as a function of analyte concentration.