Synthetic Optogenetic System to Study Bacterial Pathology

Schaefer, Rachel
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 the 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 altering 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. coli 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 the 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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