Composite
Phosphoryl

Part:BBa_K3773515:Design

Designed by: Avery Bradley   Group: iGEM21_William_and_Mary   (2021-10-14)


Circuit to report phosphorylation in vivo (alternate design)


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 324
  • 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. Our circuit consists of the ompC promoter and BBa_B0034 as an RBS driving production of sfGFP with Bba_B0015 as the terminator. Our construct builds off of the 2012 iGEM team Tokyo-NoKoGen construct (BBa_K769001); however, our team is using sfGFP instead of the original GFP.

The sequence inputted as a scar after the RBS is a spacer which has been shown to allow for expression with BBa_B0034 and sfGFP2.

UNS 1 and UNS 10 flank this part in order to allow for easy Gibson assembly as detailed by Torella et al., 20143.

This part is an improved version of BBa_K3773514.

Source

See basic parts.

References

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

2 Clifton, K. P., Jones, E. M., Paudel, S., Marken, J. P., Monette, C. E., Halleran, A. D., ... & Saha, M. S. (2018). The genetic insulator RiboJ increases expression of insulated genes. Journal of biological engineering, 12(1), 1-6.

3Torella, 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.