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Team:Dundee/Parts/Improvements
From 2013.igem.org
Reporter Gene
Osmotic stress in many Gram-negative bacteria is regulated by a two-component signal transduction system composed of EnvZ and OmpR.
EnvZ is a membrane receptor and when it is activated, by changes in osmolarity, it phosphorylates OmpR. Phosphorylated OmpR binds to DNA regulating transcription. In E. coli, this system controls the differential expression of the outer membrane porin proteins OmpF and OmpC.
To test whether our engineered EnvZ is capable of detecting microcystin, a reporter gene was constructed using three parts from the registry. This reporter consists of an OmpR binding site and ompC promoter (BBa_R0083), which is under the control of the EnvZ receptor, a strong Ribosome Binding Site (BBa_B0034) and Green Fluorescent Protein (BBa_E0040). The construct utilises GFP production to quantify EnvZ activation.
To determine whether our reporter construct responded appropriately to EnvZ activation state, we transformed the E. coli BW25113 strain (envZ+) and an isogenic envZ deletion strain with our construct. We inoculated the envZ+ and envZ- strains harbouring the construct into wells of a 96 well-plate containing growth medium supplemented with different concentrations of sodium chloride. Fluorescence was then measured after 7 hours of aerobic growth. As shown in Figure 1, the relative fluorescence varied with changes in sodium chloride concentration in the envZ+ strain but not in the envZ- strain. This shows that our OmpC-GFP reporter responds to changes in osmolarity in an EnvZ-dependent manner.
EnvZ is a membrane receptor and when it is activated, by changes in osmolarity, it phosphorylates OmpR. Phosphorylated OmpR binds to DNA regulating transcription. In E. coli, this system controls the differential expression of the outer membrane porin proteins OmpF and OmpC.
To test whether our engineered EnvZ is capable of detecting microcystin, a reporter gene was constructed using three parts from the registry. This reporter consists of an OmpR binding site and ompC promoter (BBa_R0083), which is under the control of the EnvZ receptor, a strong Ribosome Binding Site (BBa_B0034) and Green Fluorescent Protein (BBa_E0040). The construct utilises GFP production to quantify EnvZ activation.
To determine whether our reporter construct responded appropriately to EnvZ activation state, we transformed the E. coli BW25113 strain (envZ+) and an isogenic envZ deletion strain with our construct. We inoculated the envZ+ and envZ- strains harbouring the construct into wells of a 96 well-plate containing growth medium supplemented with different concentrations of sodium chloride. Fluorescence was then measured after 7 hours of aerobic growth. As shown in Figure 1, the relative fluorescence varied with changes in sodium chloride concentration in the envZ+ strain but not in the envZ- strain. This shows that our OmpC-GFP reporter responds to changes in osmolarity in an EnvZ-dependent manner.
Figure 1: The OmpC-GFP reporter responds to environmental osmolarity in an EnvZ-dependent manner. E. coli strain BW25113 (envZ+), or the isogenic envZ- mutant, expressing a plasmid-encoded ompC-gfp transcriptional fusion were cultured in media containing the indicated concentrations of sodium chloride. After seven hours, the relative fluorescence of the cultures was determined (ex. 485 nm, em. 535 nm).
