Part:BBa_K4061020

Engineered EnvZ (facilitates scaffolding)

EnvZ is a histidine kinase, part of the Two Component System in E. coli. This membrane protein is responsible for phosphotransfer to OmpR, its cognate response regulator, upon sensing changes in osmolarity within a cell. This engineered EnvZ contains the CDS of EnvZ, SH3 binding domain on its C terminus, and an auto-inhibitory domain around its four-helix bundle motif.

The SH3 binding domain facilitates scaffolding between EnvZ and OmpR which could lead to an enhanced rate of phosphotransfer. This will inturn upregulate the expression of OmpR regulated promoter, OmpC. This enhanced interaction will only be observed in the presence of the scaffold protein (BBa_K4061015).

The auto-inhibition of EnvZ prevents non-specific signal. Our project required finetuning such that the OmpF promoter (BBa_K4061000) is also activated which occurs only at low cellular osmolarity since there is less concentration of phosphorylated OmpR. In order to prevent uncontrolled upregulation of OmpC and providing a mechanism for favouring OmpF equally, we encoded an auto-inhibitory domain on EnvZ which is a SH3 ligand present in the middle of the protein near the "four helix bundle" which is the active domain responsible for phosphotransfer. The SH3 binding domain on the C terminus could bind with the SH3 ligand and this interaction can block phosphotransfer between OmpR and EnvZ. This can be reversed in the presence of scaffold, which contains an SH3 ligand of a smaller dissociation constant.

• This part is yet to be characterised, wet lab work is in progress *

Unengineered and Engineered EnvZ protein

Contribution: HKUST 2021

Summary

We ran a SWISS-MODEL to visualise our engineered EnvZ. We can clearly observe the auto-inhibition peptide around the peptide turn which acts as the kinase domain. However, the smaller peptide modifications are not evident here.

Figure 1 (a) Native EnvZ kinase protein; (b) Auto-inhibitory SH3 ligand around four-helix bundle motif

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1396
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 942
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1546