Part:BBa_K621001

OMP-R-->RBS-->eYFP

Our part consists of an OMP-R binding region that regulates our reporter gene, eYFP. The OMP-R binding region is an omp-c operator, which has three OMP-R binding sites. Our part is regulated by the ENVZ-OMPR two-component regulatory system. ENVZ is a osmolarity sensor on the inner membrane and phosphorylates OMP-R in the presence of high osmolarity. When phosphorylated, OMP-R binds to the omp-c operator and recruits RNAP to transcribe the downstream reporter gene. Fluorescence intensity due to the production of eYFP is a measure of the system's activity. When all three binding sites are occupied by phosphorylated OMP-R proteins, RNAP cannot bind to the DNA and transcribe downstream. This part can be used as an osmolarity indicator. Our team used this part as a glucose sensor because the presence of glucose changes osmolarity. Different concentrations of glucose produce different, quantifiable fluorescence intensities.

Fluorescence wavelengths

• Excitation max - 485nm
• Emission max - 520nm

Growth Conditions

• Growth Temperature and Rate - DH5alpha E.coli used as Chassis, standard growth conditions apply
• Growth Media - Nutrient Broth, since OMP-R is an osmolarity receptor the cells must be grown in a low osmolarity media (nutrient broth) to control for confounding factors.

If cells are grown in a high osmolarity media, such as LB broth, the background fluorescence will be higher than an induced glucose response. This is because the system is already primed from the high osmolarity of the media. When additional sources of osmolarity are added, the system will down regulate eYFP due the the presence of too many OMP-R proteins. Below is a dose response in the presence of LB media. Note how glucose, in these conditions, has an inhibitory effect on the system.

Dose Response

This part has been sequence verified and works as expected. In the first graph you can see that the system fluoresces over background at 1% Glucose and then drops back to background at 5% Glucose. The increase at 1% is due to positive regulation of the ENVZ-OMPR system. 5% glucose is a high enough concentration to induce downregulation of the system. Too many OMP-R proteins become phosphorylated and bind the omp-c promoter. When all three binding sites are filled RNAP cannot transcribe downstream and fluorescence decreases.

Time Trial

The graph below shows time trials of the system. These time trials were run in the presence 0%, 1%, and 8% glucose. We chose to do this so we could gather more comprehensive data. The first conclusion we can draw from this graph is that the response is fairly consistent over time (there is no peak in fluorescence associated with a specific time point). Also, for each time point between 0.5-4.5 hrs, the curve of the dose response remains consistent. This graph also shows that the assay is not accurate after 4.5 hours. The data beyond 4.5 hours becomes random and inconsistent.

Due to the nature of the system,

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]