Team:Brown/Project Histamine Sensor

To synchronize production of rEV131 with fluctuations in histamine concentration, a histamine receptor is necessary. Natural histamine receptors exist only in eukaryotes as G-coupled protein receptors, unusable for our prokaryotic cells. Therefore we have set out to engineer our own receptor. This novel receptor will sense extracellular concentrations of histamine and initiate an intracellular signal cascade that leads to the production of rEV131. rEV131 would in turn sequester histamine and lower the extracellular concentration, thus diminishing transcription of rEV131. By design, this system uses negative feedback and is self-regulating.

We approached the development of a histamine receptor with two different strategies. First, we set out to mutate the Tar periplasmic receptor domain of the Taz chimera protein. This receptor, normally sensitive is normally sensitive to Aspartate. We performed a site-directed mutagenesis of the Aspartate binding pocket. The amino acids that take part in ligand binding have been specified in the paper (Yeh, et al. 1996).

Loren Looger at Duke University used his computational protein design program to calculate mutations that would transform Tar’s aspartate binding pocket to a histamine binding pocket. His algorithm gave us the top 16 receptor designs and we are currently in the process of creating this library of mutants. We have designed primers for each of these designs and are introducing these mutations by both the “Round the Horn Site-Directed Mutagenesis” protocol on OpenWetWare and the Strategene Mutagenesis II Kit.

Our assay to test these receptors’ affinities for histamine is based on fluorescence. We have constructed a cassette from the registry that places the OmpC promoter gene over the gene for RFP. The Taz receptor, upon binding of its ligand, has an EnvZ intracellular kinase domain that phosphorylates the transcription factor OmpR, which subsequently activates transcription of the OmpC gene. With this OmpC-RFP reporter cassette, we can test both quantitatively and qualitatively the receptor’s affinity for the ligand. We have tested this signaling cascade by transforming the normal Taz receptor with the reporter cassette and introducing the ligand Aspartate to show that it works. The E. coli strain RU1012 was used, as it is an EnvZ knockout strain.

RU1012 with OmpC-RFP + Taz1 receptor

RU1012 with OmpC-RFP

RU1012 with Taz1 receptor

RU1012 with no plasmid

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Secondly, we set out to mutagenize the endogenous ribose binding protein, using our own receptor design computer program. <insert Will’s blurb about his protein design>

When a prokaryotic histamine receptor is obtained, we will place the OmpC promoter over rEV131, thus creating a self-regulating drug factory in the nose.

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