Integrative Mathematical and Experimental Approaches to Understanding Robust Activation of Gene Expression by Light Color

Indiana University, Bloomington IN

Investigators

Abstract

This research defines a newly discovered light color sensing system in bacteria that has the potential to regulate responses in synthetically created bacterial life forms. Such customized organisms have the potential to produce a wide range of beneficial products for humankind, but their activities must be strictly controlled. At present, there are few highly robust control systems available to synthetic biologists, therefore the discovery and development of additional regulatory pathways is of paramount importance. Building on recent discoveries in a highly abundant, photosynthetic marine bacterium, this project uses mathematical modeling to predict the mechanism through which shifting ambient light color from green to blue results in a 35-40-fold increase in the bacterium's gene expression. The project also involves the use of molecular, genetic, and biochemical tools to test the models and define the photo-regulation process. This system has tremendous potential for significantly contributing to the areas of microbial biotechnology, green chemistry and the new and rapidly growing field of optogenetics. In addition to the basic scientific value of the project there is training received, by three graduate students, in modeling and molecular genetics research. There is also training received, in science pedagogy, by high school teachers. Students from underrepresented groups in science benefit from a summer research immersion program at Indiana University. Mathematical modeling is used to predict the mechanism through which a newly discovered signal transduction pathway operates. Predictions from the model are tested using molecular biological and biochemical approaches. Diametrically regulated by blue and green light and widely used by Synechococcus, this simple but robust signaling system is unique. The regulation element consists of three regulatory proteins named FciA, FciB, and FciC, and three genes whose expression is highly upregulated in blue light and downregulated in green light. FciA either operates with, or independently of, FciC, and modeling these components provides weighted evidence for one of these two models. Using RNAseq and Western blot analyses a time course of RNA and protein responses is examined after a switch from growth in green light to growth in blue light. Both in vitro and in vivo approaches are used to determine the specific DNA sequences that are bound by FciA, FciB, and FciC. Methods include Electromobility Shift Assays, DNaseI footprinting, and exoChIPseq. Finally, the signaling system is transferred to another cyanobacterial species and to a non-photosynthetic bacterium to begin to develop this regulatory system for use in species that are commonly used in microbial biotechnology. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.