Engineering Systems of Anti-Repressors for Next-Generation Transcriptional Programming

The ability to control gene expression in more precise, complex, and robust ways is becoming increasingly relevant in biotechnology and medicine. Synthetic biology has sought to accomplish such higher-order gene regulation through the engineering of synthetic gene circuits, whereby a gene’s expression can be controlled via environmental, temporal, or cellular cues. A typical approach to gene regulation is through transcriptional control, using allosteric transcription factors (TFs). TFs are regulatory proteins that interact with operator DNA elements located in proximity to gene promoters to either compromise or activate transcription. For many TFs, including the ones discussed here, this interaction is modulated by binding to a small molecule ligand for which the TF evolved natural specificity and a related metabolism. This modulation can occur with two main phenotypes: a TF shows the repressor (X+) phenotype if its binding to the ligand causes it to dissociate from the DNA, allowing transcription, while a TF shows the anti-repressor (XA) phenotype if its binding to the ligand causes it to associate to the DNA, preventing transcription. While both functional phenotypes are vital components of regulatory gene networks, anti-repressors are quite rare in nature compared to repressors and thus must be engineered. We first developed a generalized workflow for engineering systems of anti-repressors from bacterial TFs in a family of transcription factors related to the ubiquitous lactose repressor (LacI), the LacI/GalR family. Using this workflow, which is based on a re-routing of the TF’s allosteric network, we engineered anti-repressors in the fructose repressor (anti-FruR – responsive to fructose-1,6-phosphate) and ribose repressor (anti-RbsR – responsive to D-ribose) scaffolds, to complement XA TFs engineered previously in the LacI scaffold (anti-LacI – responsive to IPTG). Engineered TFs were then conferred with alternate DNA binding. To demonstrate their utility in synthetic gene circuits, systems of engineered TFs were then deployed to construct transcriptional programs, achieving all of the NOT-oriented Boolean logical operations – NOT, NOR, NAND, and XNOR – in addition to BUFFER and AND. Notably, our gene circuits built using anti-repressors are far simpler in design and, therefore, exert decreased burden on the chassis cells compared to the state-of-the-art as anti-repressors represent compressed logical operations (gates). Further, we extended this workflow to engineer ligand specificity in addition to regulatory phenotype. Performing the engineering workflow with a fourth member of the LacI/GalR family, the galactose isorepressor (GalS – naturally responsive to D-fucose), we engineered IPTG-responsive repressor and anti-repressor GalS mutants in addition to a D-fucose responsive anti-GalS TF. These engineered TFs were then used to create BANDPASS and BANDSTOP biological signal processing filters, themselves compressed compared to the state-of-the-art, and open-loop control systems. These provided facile methods for dynamic turning ‘ON’ and ‘OFF’ of genes in continuous growth in real time. This presents a general advance in gene regulation, moving beyond simple inducible promoters. We then demonstrated the capabilities of our engineered TFs to function in combinatorial logic using a layered logic approach, which currently stands as the state-of-the art. Using our anti-repressors in layered logic had the advantage of reducing cellular metabolic burden, as we were able to create the fundamental NOT/NOR operations with fewer genetic parts. Additionally, we created more TFs to use in layered logic approaches to prevent cellular cross-talk and minimize the number of TFs necessary to create these gene circuits. Here we demonstrated the successful deployment of our XA-built NOR gate system to create the BUFFER, NOT, NOR, OR, AND, and NAND gates. The work presented here describes a workflow for engineering (i) allosteric phenotype, (ii) ligand selectivity, and (iii) DNA specificity in allosteric transcription factors. The products of the workflow themselves serve as vital tools for the construction of next-generation synthetic gene circuits and genetic regulatory devices. Further, from the products of the workflow presented here, certain design heuristics can be gleaned, which should better facilitate the design of allosteric TFs in the future, moving toward a semi-rational engineering approach. Additionally, the work presented here outlines a transcriptional programming structure and metrology which can be broadly adapted and scaled for future applications and expansion. Consequently, this thesis presents a means for advanced control of gene expression, with promise to have long-reaching implications in the future.

Date

2021-08-13

Resource Type

Text

Resource Subtype

Dissertation

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