DESIGN AND CONSTRUCTION OF GENETIC CIRCUITS FOR ENGINEERING METHYL SELECTIVE DNA BINDING DOMAINS

Abstract

DNA methylation is a crucial epigenetic modification that modifies protein-DNA interactions and regulates many essential processes in prokaryotes and eukaryotes. Control over DNA methylation has applications in disease characterization and treatment, memory-based genetic circuits, and cellular reprogramming. However, current approaches to studying and modifying DNA methylation lack the ability to selectively target methylated sequence-specific DNA. Furthermore, the conventional protein engineering workflow for designing DNA-binding proteins with methyl-selective, sequence-specific DNA recognition relies on inefficient multi-step screening processes. To address these limitations, a genetic circuit is proposed as a high-throughput selection platform for engineering sequence-specific, methylation-selective DNA-binding domains. The pCD plasmid was designed to present a helix-turn-helix repressor library with both methylated and unmethylated operators, and to integrate methylation-selective DNA recognition into an output that could be interrogated by GFP screening and kanamycin resistance selection. The pCD plasmid was successfully constructed using PCR-based DNA assembly of modular circuit components. Kanamycin growth results supported the use of kanamycin resistance as a selection marker to evaluate the binding specificity of engineered DNA-binding domains. The initial failure to establish circuit controls suggested the incorporation of a non-functional repressor construct, Rz. A simplified plasmid, pAD.02, was designed to further investigate the interaction between RZ and the corresponding operator, ZZ. RZ variants, XSN, XAN, XKR, and KSX were paired with TTA, TGT, GAC, and CGG operators, respectively, based on previously characterized LacI DNA-binding-domain operator interactions. These interactions were assessed across the proximal, core, and distal operator positions and revealed that the established LacI DNA-binding domain-operator pairings are not fully compatible within the RZ architecture. In total, 14 functional RZ-ZZ operator pairings were identified and can be incorporated as regulatory components in pCD. Overall, this work advances the development of a genetic circuit-based screening and selection platform for engineering methyl-selective DNA-binding proteins and suggests that surrounding structural features may influence DNA binding domain-operator interactions.