Ph.D Thesis


Ph.D StudentLitovco Phyana
SubjectTopologies of Synthetic Gene Circuit for Optimal
Fold Change Activation
DepartmentDepartment of Biomedical Engineering
Supervisor ASSOCIATE PROF. Ramez Daniel
Full Thesis textFull thesis text - English Version


Abstract

Synthetic biology aims to reprogram living cells to control cellular signal processing for redirected, practicable applications. In this work, we demonstrate the design and fabrication of whole-cell biosensors based on Escherichia coli cells implementing various engineering concepts adapted from synthetic biology.

To date, the main guideline in whole-cell biosensors engineering has mainly based on single-input, encompassing a promoter which senses the presence of the toxin, activating and followed by a second unit, which is a reporting gene. Various whole-cell bacterial strains based on single input have been genetically engineered to detect a broad spectrum of toxic chemicals such as those that cause DNA damage, membrane damage, protein damage, and oxidative stress. However, such concept has greatly failed to deal with real-world samples which require higher computational complexity, low detection threshold, specificity, and stability.

Computations widely exist in biological organisms for sensing and responding to various stimuli inside or/and outside the cells. These computations are assembled by genetic circuits. In this work we engineered genetic transcriptional regulatory circuits responsible for biosensing function, by integration of feedforward and feedback loops. Although, feedforward and feedback loops naturally exist in the transcriptional regulation networks in various signaling pathways, in this work we built synthetic loops i.e., that don’t naturally exist in the living cells.

Fold-change activation (FCA) in gene regulatory networks is defined as the ratio between the ON state, when an activated promoter has maximum activity, and the OFF state (basal level), when an activated promoter has minimum activity, which is produced when RNA polymerases bind to the promoter in the absence of stimulus The basal level activity and FCA levels of the promoter play a significant role in determining the behavior of gene circuits in terms of sensitivity, precision and accuracy, dynamic range, noise filtering and more.

In this work, we built three model-guided designs: open loop (OL), indirect coherent feedforward (ICF) loop and double negative feedback (DNF) loop or mutual inhibition, combining feedforward and feedback loops. ICF and DNF designs include an interference component formed by either transcriptional interference or antisense transcription and a repressor that can control those interferences. We demonstrated that an ICF and DNF designs can experimentally improve the fold change response of promoters, by reducing the basal level as much as possible without compromising the maximum activity level. OL, ICF and DNF designs have been applied first on synthetic inducible promoters as a proof-of-principle and then have been applied on native promoters that are either functionally specific or systemically involved in complex repair pathways in living cells such as oxidative stress and SOS response. In all six promoters, an improvement of up to ten times in the level of fold change activation was observed. We expect that this methodology can be applied in various biological systems for biotechnology and therapeutic applications