Part:BBa_K3773514:Design

Circuit to report phosphorylation in vivo

Assembly Compatibility:

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

  Illegal AgeI site found at 330
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This circuit utilizes the EnvZ and OmpR system found natively in E. coli. EnvZ is an outer membrane protein that responds to changes in the osmolarity of the surrounding environment. When this protein detects osmolarity changes, it auto-phosphorylates then passes the phosphate to the OmpR protein. Depending on the osmolarity of the surrounding medium, OmpR will either bind to the OmpC promoter in high osmolarity states or to the OmpF promoter in low osmolarity states, producing these genes coding for additional outer membrane proteins¹. BBa_K3773515 is an improved version of this part; it builds off of the 2012 iGEM team Tokyo-NoKoGen construct (BBa_K769001). Unlike BBa_K3773515, BBa_K3773514's promoter and RBS sequence are obtained from Schmidl et al., 2014.

UNS 1 and UNS 10 flank this part in order to allow for easy Gibson assembly as detailed by Torella et al., 2014².

Source

Schmidl, S. R., Sheth, R. U., Wu, A., & Tabor, J. J. (2014). Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS synthetic biology, 3(11), 820-831.

References

¹ Cai, S. J., & Inouye, M. (2002). EnvZ-OmpR interaction and osmoregulation in Escherichia coli. Journal of Biological Chemistry, 277(27), 24155-24161.

²Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2014). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, 42(1), 681-689.