IDEALS Home University of Illinois at Urbana-Champaign logo The Alma Mater The Main Quad

Transcription Factor Engineering: Tools and Applications

Show full item record

Bookmark or cite this item: http://hdl.handle.net/2142/18624

Files in this item

File Description Format
PDF McLachlan_Michael.pdf (3MB) (no description provided) PDF
Title: Transcription Factor Engineering: Tools and Applications
Author(s): McLachlan, Michael J.
Director of Research: Zhao, Huimin
Doctoral Committee Chair(s): Zhao, Huimin
Doctoral Committee Member(s): Katzenellenbogen, John A.; Zhong, Sheng; Gerlt, John A.
Department / Program: School of Molecular & Cell Bio
Discipline: Biophysics & Computnl Biology
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Transcription factor estrogen receptor alpha protein engineering biosensor vascular endothelial growth factor-A (VEGF-A)
Abstract: Transcription factors play a vital role in the biology of every organism. By controlling gene expression they regulate growth, development, metabolism, reproduction, signaling, and response to the environment. They have also provided the basis for many useful tools in molecular biology. The estrogen receptor alpha is one of the most studied human transcription factors and acts as a ligand controlled regulator of transcription. The modular design of this and other transcription factors allows for the rational design of artificial gene switches to control expression of desired genes. In this thesis I explore some tools for, and applications of, engineering the estrogen receptor. Beginning with two ligand binding domain mutants previously engineered to recognize the small molecules 4,4’-dihydroxybenzil (DHB) or 2,4-di(4-hydroxyphenyl)-5-ethylthiazole (L9), I showed that they could be used as a gene switch to independently control reporter genes in yeast and mammalian cells. By using different DNA binding domains, activation and repression domains, promoter elements, and a luciferase reporter I implemented the logic functions AND, OR, NAND, and NOR in HeLa cell culture. My research revealed some of the limitations of both the modular engineering approach, and the yeast two-hybrid screening assay used to engineer the ligand binding domain. I explored the feasibility of performing directed evolution of gene switches in mammalian cells through a protoplast fusion method, which combines the benefits of simple library creation with screening in a functionally relevant system. Although individual steps of the process were successful, the method proved unsuitable for large scale screening of libraries. The endogenous gene vascular endothelial growth factor-A (VEGF-A) was targeted for control by a gene switch. The effect of construct design was evaluated using a VEGF-A promoter controlled luciferase gene and performance was impacted by the choice of DNA binding domain, activation domain, and the order of domain use. Endogenous VEGF-A protein secretion in HeLa cells was successfully upregulated twofold by a DHB ligand controlled gene switch. Finally, I developed a useful biosensor for estrogenic compound detection that has the advantage of requiring no added substrates for signal generation. Through fusing the N and C terminal halves of the fluorescent protein Venus to the receptor ligand binding domain, fluorescence complementation generated a robust signal upon addition of an estrogenic ligand. The biosensor was capable of responding to a range of endogenous, pharmaceutical, environmental, and industrial compounds with sensitivities that correlated with their relative binding affinity. The signal characteristics were seen to depend on the length of the LBD region used, with some constructs distinguishing between agonists and antagonistic ligands.
Issue Date: 2011-01-21
URI: http://hdl.handle.net/2142/18624
Rights Information: Copyright 2010 Michael McLachlan
Date Available in IDEALS: 2011-01-21
2013-01-22
Date Deposited: 2010-12
 

This item appears in the following Collection(s)

Show full item record

Item Statistics

  • Total Downloads: 182
  • Downloads this Month: 11
  • Downloads Today: 0

Browse

My Account

Information

Access Key