Dr. Tina Lebar received Pregl award for PhD thesis
Dr. Tina Lebar received Pregl award for PhD thesis »Designing gene regulatory circuits based on DNA binding proteins«, which she defended in July 2017. The research was conducted under supervision of prof. dr. Mojca Benčina.
Abstract of thesis: Gene regulatory circuits are composed of molecular elements which control cellular processes depending on the presence of different chemical or physical signals, in order to determine cell behaviour. By implementing rational, controllable elements into living cells, we can apply such »biological machines« for different functions. Most synthetic biological systems, implemented up to now, utilizes natural bacterial regulatory elements. Such bacterial transcription factors are available in limited numbers, while they are also involved in regulation of cellular processes and interact with other molecules in cells. Due to their diverse biochemical properties their behaviour in complex systems is unpredictable, additionally limiting their application for building complex biological circuits. In this doctoral dissertation, we use transcription factors based on modular, designed DNA binding proteins for construction of synthetic gene regulatory circuits. Such transcription factors can be synthesized in unlimited numbers and designed to bind any DNA sequence, while maintaining their original biochemical properties. Initially we compared transcription factors based on TAL effectors (»Transcription Activator-Like effectors«) and the CRISPR system ( »Clustered Regularly Interspaced Short Palindromic Repeats«), where TAL effectors were shown to be more efficient. We prepared transcriptional repressors and activators based on both systems and improved their activation efficiency by using coiled-coil peptides. We prepared improved variants of TAL effector-based transcriptional activators, responsive to blue light illumination. We demonstrate the versatility of TAL effector-based elements for construction of complex logic in mammalian cells by construction of a genetic bistable toggle switch and a genetic oscillator. At the same time, we utilize the unique binding mechanism of TAL effectors for introduction of a new mode of transcriptional inhibition by directed displacement of trascriptional activators, which we use to implement all 14 non-trivial two-input logic gates and a half-adder circuit in mammalian cells. Additionally, we demonstrate the wider use of TAL effector-based directed displacement by inhibition of Cas9 endonuclease-mediated DNA cleavage, enabling the use of TAL effectors for DNA protection from off-target cleavage when using the CRISPR system for genome editing. Modular, designed DNA binding proteins facilitate construction of complex circuits with a large number of elements, while they also enable building different, orthogonal circuits in a single cell. Cells, designed to carry out complex tasks, for example data storage or performance of logic functions, will potentially be useful for biotechnological and biomedical applications, where »programmed« cell behaviour is desired. Designed transcription factors based on TAL effectors and other platforms represent a new level of standardization in synthetic biology and will allow for an almost unlimited complexity of synthetic circuits.