Project

Abstract

In the bodies of people with diabetes, the ability to recognize and respond to glucose concentrations in the blood has been compromised. As a result, glucose accumulates to dangerous levels. High blood glucose concentrations can cause irreversible damage to critical organs, impairing their functionality. With parts from the iGEM registry, our team created a glucose-controlled promoter linked to a yellow fluorescence production gene in E. coli. The concentrations of glucose to which the promoter responds can be determined. Once the concentration is known, the promoter can be mutated so that it will be activated by varying concentrations of glucose and be used as a glucose sensor for people with diabetes. In the future, an insulin gene could be added to this system for use in insulin pumps, where specific glucose levels trigger insulin production in E. coli.

Our System Overview

We submitted two biobricks to the registry: an intermediate part (http://partsregistry.org/wiki/index.php?title=Part:BBa_K621000) with a ribosome binding site and eYFP, and the intermediate plus the ompC operator (http://partsregistry.org/wiki/index.php?title=Part:BBa_K621001). The ompC operator has three binding sites for phosphorylated OmpR. OmpR and EnvZ work together in a two-component regulatory system as shown in the diagram below.

EnvZ is an inner membrane protein that senses osmolarity. Phosphorlyation of OmpR by EnvZ positively correlates with the osmolarity of the system. Phosphorylated OmpR (OmpR-P) can bind to the three sites on the ompC operator. When one or two of the binding sites are occupied by OmpR-P, RNA polymerase is recruited to begin downstream transcription of the reporter gene, eYFP. However, when all three OmpR binding sites are occupied by OmpR-P, RNA polymerase cannot bind, the reporter gene can no longer be produced, and therefore the system is inhibited. In summary, as osmolarity increases from very low levels, the fluorescence produced by the system also increases until the system reaches a threshold osmolarity. Once the system reaches the threshold, the fluorescence will decrease with increasing osmolarity due to the inherent down-regulation of the system. The activity of the system can be quantified using the fluorescence of the cells because the two-component regulatory system of EnvZ and OmpR regulates transcription of the eYFP gene, dictating the level of fluorescence.

Making Our Parts

We started with three parts from the iGEM registry:

• BBa_B0032: RBS-3, a medium-strength ribosome binding site
• BBa_E0030: eYFP gene, codes for yellow fluorescence
• BBa_R0082: omp-c operon, contains three binding sites for phosphorylated OmpR

Testing

Our team performed multiple assays with 96-well plates. See data page.

Next Steps

Mutagenesis.