Mapping interactions between metabolites and transcriptional regulators at a genome-scale
The control and regulation of cellular metabolism is required to maintain the biosynthesis of building blocks and energy, but also to prevent the loss of energy and to be able to quickly adjust to changing conditions. Hence, the metabolic network and the flow of genetic information has multiple laye...
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|Summary:||The control and regulation of cellular metabolism is required to maintain the biosynthesis of building blocks and energy, but also to prevent the loss of energy and to be able to quickly adjust to changing conditions. Hence, the metabolic network and the flow of genetic information has multiple layers of regulation and information is transmitted between gene expression and metabolism. For this purpose, metabolites serve as key signals of the regulatory network to balance metabolism via the adjustment of protein levels and the activity of enzymes. Understanding these regulations and interplays of bacterial metabolism will enable us to improve the modelling and engineering of metabolic networks and ultimately to develop new antibiotics and production strains. The aim of this thesis is to investigate which regulatory mechanisms are used by the cell to respond to genetic perturbations. Moreover, we develop new methods to map protein-metabolite interactions and to prove their functionality in the cell.
After introducing the fundamentals of metabolic network regulation, we investigate in chapter 1 how Escherichia coli (E. coli) reacts to genetic perturbations. We use a library of 7177 CRISPRi strains to perform a pooled fitness growth assay, demonstrating the buffering effects of metabolism. Additionally, measuring the metabolome and proteome of 30 arrayed CRISPRi strains enables us to elucidate three gene-specific buffering mechanisms.
In chapter 2, we use our new insights about genetic perturbations of chapter 1 to develop a method for systematically mapping interactions between metabolites and transcriptional regulators. CRISPRi leads to a knockdown of a gene and therefore induces specific changes in the metabolome and proteome of the cell. We therefore combine the pooled CRISPRi library with a fluorescent reporter for transcription factor activity and extract cells, which show a response of the reporter to the changing conditions, via FACS from the pooled library. By analyzing proteome and metabolome data, we confirm previously reported and discover new interactions.
With chapter 3, we provide a detailed protocol of how to work with CRISPRi libraries. We explain the design and construction of sgRNAs of arrayed as well as pooled CRISPRi strains and how to perform growth assays. Furthermore, we explain the execution and analysis of Illumina Next-generation sequencing of pooled libraries. We also explain the sorting of cells from pooled libraries via FACS.
In chapter 4, we show how to find new interactions between metabolites and transcription factors by external perturbations. By switching a growing E. coli culture between growth and glucose limitation, we provoke strong changes of metabolite levels and transcript levels. Calculating the transcription factor activity from gene expression levels and correlating them with metabolite levels, enables us to recover known interactions but also to discover new interactions, of which we prove five in in vitro binding assays.
In chapter 5, we investigate the function of allosteric regulation of metabolic enzymes in amino acid pathways of E. coli. We constructed 7 mutants of allosteric enzymes to remove the allosteric feedback regulation. By metabolomics, proteomics and flux profiling analysis we show how allostery helps to adjust enzyme levels of the cell. Furthermore, using a metabolic model and the application of CRISPRi we show how well-adjusted enzyme levels make the cell more stable towards genetic perturbations.|
|Physical Description:||209 Pages|