Student Theses and Dissertations

Author

Sergio Botero

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Simon Laboratory

Abstract

Phosphoinositides are membrane phospholipids involved in a wide variety of processes across the tree of life. In eukaryotic cells they function though their role as integral membrane components, anchors for proteins, membrane identity markers, and signaling molecules. Phosphoinositides are regulated through the phosphorylation and dephosphorylation of the inositol head-group at the 3rd, 4th, and 5th positions, creating a complex and very dynamic interconversion network. They can also be hydrolyzed into an inositide head-group and diacylglycerol which are in turn signaling molecules. The wide variety of functions, and redundancy in their synthesis pathways, makes the in-vivo study of phosphoinositides complex since any experimental alterations can have undesired effects throughout the cell. In this work I engineered the metabolic network required to synthesize the most abundant eukaryotic phosphoinositides in the bacterium Escherichia coli, which normally lacks any of these phospholipids. This engineered bacterium is a new tool for the in-vivo study of cell biology models that involve phosphoinositides, allowing for a precise control of the system and avoiding any undesired interactions. To achieve this I built and optimized the expression of the required enzymes into a single plasmid such that it can be used in any strain of E. coli. My system can produce phosphatidyl inositol, phosphatidyl inositol (4) phosphate (PI4P), and phosphatidyl inositol (4,5) diphosphate (PIP2), and is easily controlled trough the addition of inositol to the growth media of the bacterium. As an example application of my system, I use it to confirm the role of PIP2 binding in the non-conventional protein export of human basic fibroblast growth factor (FGF2).

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

Available for download on Saturday, April 28, 2018

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