Arkady B. Khodursky - US grants

The overall objective of this project is to determine the relationship between phenotypic variations and the associated whole genome transcriptional pattern in populations of E.coli and S. cerevisiae cells cultivated under well defined, reproducible conditions. This will be done in three types of experiments distinguished according to the cultivation mode. The first type of experiments will involve simple batch cultivation of cells. Samples will be taken from different growth stages of the culture. The second type of experiments will involve continuous cultivation of cells in a chemostat operated at different steady states. The third type of experiments will involve exposure of the cells to an environmental challenge. Such challenges will include the shifting of a steady state chemostat culture to a new dilution rate. The response to such a nutritional upshift or downshift will be analyzed. In all types of experiments the phenotypic variability of the culture will be determined by flow cytometry and subpopulations of cells will be isolated via cell sorting. Sorted cells will be subjected to protocols for microarray analysis to determine the whole genome transcriptional profile. To make this possible an amplification protocol will be developed to reproducibly generate a sufficient amount of RNA from only a few cells to permit quantitative microarray analysis.

DESCRIPTION (provided by applicant): Information stored in genes is realized through the process of transcription. While transcription of individual genes is understood as a biochemical reaction in a great deal of molecular detail, the transcription of gene ensembles cannot yet be studied in a framework of the reduced biochemical reaction. It is also likely that even if we were equipped to study ensemble activities with high molecular precision, we would miss essential macroscopic properties of the processes that govern transcription of gene ensembles in the cell. The most basic and fundamental is the process of transcription as a function of position of the genes on a chromosome. The fundamental nature of it can be underscored by the notion that at the deterministic basis of this process lays the structure of a chromosome - a critical piece of knowledge about the cell. Indeed, the relationship between structure and function is one of the fundamental principles in molecular biology. The relationship between molecular structure and function has been used very successfully to propose, understand, and verify mechanisms of action of individual protein and DNA molecules as well as their motifs. However, our understanding of organization of the higher order macromolecules, such as chromosomes, has been slow and ineffective largely due to the low-resolution capacity of indirect techniques and invasive nature of the direct ones. Whole genome DNA microarrays designed using complete sequence information made possible direct read-out of genome's activity at a single gene resolution and higher. My laboratory uses this technique to: i) directly study and model the structure of the Escherichia coli K12 (E. coli) chromosome; ii) determine how the structure of the chromosome influences its activity and vice versa. We demonstrated that variations in gene activity as a function of gene position contain useful information about the process of transcription and are not entirely random: significant short- (~ 5 kb) and long-range correlations (~ 90kb) can be detected in transcriptional spatial data series by using standard analytical tools borrowed from signal processing, information theory and statistics. I propose to extend the combined use of theoretical approaches with direct experimentation to determine: 1) the effects of internal and external perturbations, which are known to affect global chromosomal state(s), on the observed spatial correlations; 2) dynamic distribution of DNA biding proteins that are known to control global DNA properties; 3) 3-D structure of the bacterial chromosome. As a result of these studies I expect to be able to model mechanistic and structural basis for the chromosome activity. This work will reveal a new level of organization of prokaryotic genetic material and its role in bacterial physiology, and also will provide a framework for studying spatial interactions in the chromosomes of higher organisms.

This INSPIRE award is partially funded by the Systems and Synthetic Biology Program and the Cellular Dynamics and Function Program in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Science; and the Biophotonics Program and Biomedical Engineering Program in the Division of Chemical, Bioengineering, Environmental, and Transport System in the Directorate for Engineering.
Intellectual Merit: In this INSPIRE project, unique concepts from several disciplines are combined in a novel way to give a consistent theoretical framework for the analysis, design, construction and evolution of metabolic networks. First, the proposed work builds on the premise that metabolic networks can be discretized into fundamental pathways or elementary modes. Cells function only according to these elementary modes or combinations of modes in compliance with the mass conservation law. These unique, discrete metabolic states of a network can be rigorously evaluated with computational tools to reveal all the possibilities that a cell can perform. Because the entire property space of a cell is known, the design of a metabolic network can be carried out on a completely rational basis and then implemented with molecular biology techniques. Second, the Maximum Entropy Production principle (MEP) predicts that the most probable metabolic state is reached when the usage of the individual, discrete elementary modes is distributed according to the Boltzmann distribution law. Third, when the predictable most probable state, which is computed from the distributed usage of elementary modes, is compared with the current measurable state of the network, it is possible to predict what reaction will be altered during the course of evolution. Thus, this research has far reaching implications and, therefore, requires careful investigation to prove its general validity. If proven to be valid, this represents a design principle in synthetic biology that needs to be followed in order to engineer robust, stable systems that do not evolve further in time.
Broader Impacts: This research has the potential to transform the field of metabolic engineering in several significant ways. For example, biotechnology processes that produce valuable chemicals, including pharmaceuticals or biofuels, may be enhanced. The proposed study could also provide a platform for the sequestration and transformation of carbon dioxide into isobutanol, the production of carotenoids and bioethanol from biomass. It would further impact society by providing advanced training to the next generation of scientists, including high school, undergraduates, graduate, and post-graduate students. Most importantly, the project would lay the foundation for the scientific community to further advance and apply these groundbreaking principles in novel ways.