Part:BBa_K957001

Arsenic inducible mtrB

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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

To test BBa_K957001 we cloned mRFP downstream, and measured fluorescence while varying concentration of arsenic-containing compounds in growth medium. All trials were run with the same controls: blank LB medium, S. oneidensis with mtrB knocked out, S. oneidensis conjugated with an Anderson promoter (0.1, 0.4, and 1.0) and mRFP, and an arsenic reporter strain without mRFP appended after mtrB. Background fluorescence from LB was subtracted, and fluorescence normalized to optical density in order to obtain relative fluorescence per cell mass. Additionally, as described above in the control experiment, fluorescence data was averaged over a time course of 4.5 hours, after the cells had grown to a steady OD.

Preliminary data of relative fluorescence at 0, 10, 50, 100, and 500 μM of arsenite. Four strains of S. oneidensis are plotted: with mtrB knocked out, with an arsenic reporter without mRFP appended after mtrB, and with two arsenic reporters with mRFP appended. Relative fluorescence is reported after normalization to optical density. For the two testing strains (arsenic reporter w/ BamHI and arsenic reporter w/o BamHI), fluorescence is averaged over 3 replicates, in addition to being averaged for each replicate individually over a time course of 4.5 hours.

Preliminary data of relative fluorescence at 0, 10, 100, and 500 μM of arsenate. Four strains of S. oneidensis are plotted: with mtrB knocked out, with an arsenic reporter without mRFP appended after mtrB, and with two arsenic reporters with mRFP appended. Relative fluorescence is reported after normalization to optical density. For the two testing strains (arsenic reporter w/ BamHI and arsenic reporter w/o BamHI), fluorescence is averaged over 3 replicates, in addition to being averaged for each replicate individually over a time course of 4.5 hours.