Part:BBa_K1012005
GFP under control of an OmpR-responsive promoter
This is a composite part (a reporter) designed to respond to EnvZ activity in the E. coli chassis organism. It can be used as a biosensor for external osmolarity.
Usage and Biology
The E. coli EnvZ/OmpR two component system is designed to respond to osmotic stress (Siryaporn & Goulian 2008). EnvZ is a membrane-bound sensor histidine kinase. Increased osmolarity activates the kinase which autophosphorylates itself. The phosphate group is then picked up by OmpR, a cytoplasmic response regulator, which then promotes transcription of the ompC gene. OmpC is an outer membrane protein that is abundant under conditions of high osmolarity.
This new BBa_K1012005 device is composite part of the ompC promoter/operator (BBa_R0083) linked via a ribosome binding site (BBa_B0034) to GFP (BBa_E0040).
The immediate aim in 2013 was for the Dundee 2013 iGEM team to engineer EnvZ to respond to different environmental signals other than osmolarity. Then, it was hypothesised, the BBa_K1012005 device could be used to report the presence of other signalling molecules in the environment. First, the function of the new BBa_K1012005 device had to be characterised and quantified in relation to native EnvZ activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 748
Functional Parameters
To determine whether the BBa_K1012005 reporter device responded appropriately to EnvZ activation state, the E. coli BW25113 strain (which is envZ+) and the isogenic envZ deletion strain JW3367 from the Keio Collection (curated at the University of Dundee) were chosen as chassis. Both strains were transformed separately with BBa_K1012005 and cultured in rich medium. Next, the envZ+ and envZ- strains harbouring the BBa_K1012005 reporter were inoculated the into wells of a 96 well-plate containing growth medium supplemented with different concentrations of sodium chloride. GFP fluorescence was then measured after 7 hours of aerobic growth. As shown in Figure 1, the relative GFP fluorescence increased and then stabilised with increasing external sodium chloride concentration in the envZ+ strain. However, when the BBa_K1012005 reporter was expressed in the envZ mutant strain no induction of GFP fluoresence was observed (Figure 1). This clearly demonstrates that the BBa_K1012005 reporter device is functional and the levels of GFP fluorescence allow some quantification of EnvZ/OmpR activity.
Figure 1: The BBa_K1012005 reporter responds to environmental osmolarity in an EnvZ-dependent manner. E. coli strain BW25113 (envZ+), or the isogenic envZ- mutant JW3367, were transformed with pSC13C carrying the BBa_K1012005 reporter and cultured in media containing the indicated concentrations of sodium chloride. After seven hours, the relative GFP fluorescence of the cultures was determined (ex. 485 nm, em. 535 nm).
| n/a | GFP under control of an OmpR-responsive promoter |
Acknowledgement of Sources
The BW25113 and JW3367 E. coli strains were provided by the Division of Molecular Microbiology, College of Life Sciences, University of Dundee.
References
Siryaporn A & Goulian M. (2008) Cross-talk suppression between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli. Molecular Microbiology 70:494-506.
| n/a | GFP under control of an OmpR-responsive promoter |