Part:BBa_M50087:Design

pH sensor-reporter device in E. coli

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2264
  Illegal BsaI.rc site found at 2748
  

Design Notes

Sensor plasmid: The sequences for ArsS and ArsR were codon optimized for use in E. coli. Additionally, we added a FLAG tag to the end of the ArsS sequence and a 6xHistidine tag to the end of the ArsR sequence so that we could easily distinguish those proteins should we perform assays such as Western Blots or ELISAs. Neither DNA sequence had a stop codon initially, so we also added the sequence "TAA" to the end of each sequence (after the tag added to each).

Reporter plasmid: We used the pUreA promoter native to Helicobacter pylori, as it has a binding site for ArsR, and it is unknown whether any promoters native to E. coli have such a binding site. We made no changes to pUreA since we are unsure as to how it may function in E. coli and wanted to increase the likelihood of it functioning properly (as it does in H. pylori).

Source

ArsS and ArsR sequences: The H. pylori ArsS and ArsR gene sequences were both obtained using UniProt and then optimized for use in E. coli using Integrated DNA Technologies’ Codon Optimization Tool. The codes in the UniProt databases for ArsS and ArsR are A0A1U9ISA2 and A0A1U9IS85, respectively.

pUreA sequence: The gene sequence for the promoter was sourced from a paper titled “Activation of Helicobacter pylori ureA promoter by a hybrid Escherichia coli-H. pylori ropD gene in E. coli.”

References

Marcus E, et al. 2012. “Role of the Helicobacter pylori Sensor Kinase ArsS in Protein Trafficking and Acid Acclimation.” Journal of Bacteriology. Vol. 194: 5545-5551.

http://www.uniprot.org/uniprot/A0A1U9ISA2

http://www.uniprot.org/uniprot/A0A1U9IS85