Part:BBa_K4767007:Design

Pars-RBS-arsR-RBS-mtrC-TT

Assembly Compatibility:

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

  Illegal BglII site found at 270
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 751
  

Design Notes

We relied on an mtrC complementation strategy to construct arsenic-responsive genetic circuits in plasmids. MtrC plays an essential role in the metal reduction (Mtr) pathway of S. oneidensis MR-1, stabilizing a complex formed with MtrA and MtrB. Because this stable complex is required for electrode reduction, strains deficient in the mtrC coding sequence are unable to produce significant levels of current when inoculated into BESs. However, when mtrC is re-introduced into a knockout strain, the electrode reduction phenotype is restored. When inoculated into a BES, current production will increase in response to increasing mtrC transcription in such an engineered strain. We exploited this by placing the mtrC coding sequence under the control of an arsenic-inducible promoter region. 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. In this design，ArsR acts as a negative auto-regulator by limiting its own expression to a low, basal level. 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, MtrC levels—and hence, electrode reduction capacities—increase with increasing arsenic presence. We also applied to another c-Cyts gene, cymA, as the reporter gene to response arsenic.

Gene circuit we designed for arsenic response

Source

This part is from S. oneidensis and Escherichia coli, assembled by BBa_K4767001, BBa_J34801 , BBa_J15101 , BBa_B015 and BBa_K3102020.

References

Xiaoqiang Jia, Bu Rongrong, Zhao Tingting, et al. Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection[J]. Applied and environmental microbiology, 2019, 85(11): 1.