Department

Molecular Biology Department

First Advisor

Dr. Mark Gomelsky

Description

A near infrared light activated system based on light - activated cGMP synthesis will provide a tool to study bacterial pathology within a mammalian host. Activation or inactivation of proteins with light will allow scientists to have precise spatiotemporal control of cellular processes and will yield great insights in to th e mechanisms of cyclic nucleotide dependent pathways. This NIR light activated system will be comprised of two pieces, a transcriptional activator and a cyclase. We have attempted to create a transcription activator which is activated by cGMP binding by a ltering the substrate specificity of E. coli transcription activator from cAMP to cGMP. Using Fusion PCR we created a chimeric protein of a cGMP specific transcription activator N terminal domain from R. centenum fused to the C terminal domain of the E. co li transcription activator. We further preformed Random PCR Mutagenesis on this fusion to obtain a functional fusion protein. Despite our best efforts we were unable to find a functioning chimeric transcription activator. Recent studies have shown that th e R. centenum cGMP specific transcription activator will activate transcription in E. coli , therefore a cGMP specific transcription activator is no longer needed. Future studies will focus on creating a NIR light - activated guanylyl cyclase.

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Synthetic Optogenetic System to Study Bacterial P athology

A near infrared light activated system based on light - activated cGMP synthesis will provide a tool to study bacterial pathology within a mammalian host. Activation or inactivation of proteins with light will allow scientists to have precise spatiotemporal control of cellular processes and will yield great insights in to th e mechanisms of cyclic nucleotide dependent pathways. This NIR light activated system will be comprised of two pieces, a transcriptional activator and a cyclase. We have attempted to create a transcription activator which is activated by cGMP binding by a ltering the substrate specificity of E. coli transcription activator from cAMP to cGMP. Using Fusion PCR we created a chimeric protein of a cGMP specific transcription activator N terminal domain from R. centenum fused to the C terminal domain of the E. co li transcription activator. We further preformed Random PCR Mutagenesis on this fusion to obtain a functional fusion protein. Despite our best efforts we were unable to find a functioning chimeric transcription activator. Recent studies have shown that th e R. centenum cGMP specific transcription activator will activate transcription in E. coli , therefore a cGMP specific transcription activator is no longer needed. Future studies will focus on creating a NIR light - activated guanylyl cyclase.