Synthetic Control of the Biological Central Dogma

Author: Gerber, Bryan Michael

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Wang, Kaihang

Committee Members: Murray, Richard M.; Newman, Dianne K.; Shapiro, Mikhail G.; Wang, Kaihang

Option: Biology

Abstract

The Central Dogma of biology dictates the flow of information within all living organisms. By focusing on the construction of synthetic DNA, the transcription of target RNA, and the translation of select proteins, we can improve the synthetic control of engineered organisms. Prioritizing the mis-connection rate present when attaching two DNA molecules, I propose two conceptual improvements to DNA assembly technologies. The high efficiency of the first technique, called Sidewinder, is demonstrated through the construction of a GFP mutation library from DNA oligos, whose diversity is confirmed by high-fidelity sequencing and functional phenotypic analysis. The second DNA assembly technique, High Temperature (HighT) assembly, is demonstrated by high efficiency plasmid ligation, direct integration into bacterial genomes by two independent recombinase HighT assembly-to-integration systems, and the construction of multiple eukaryotic genes including MAPT, VEGFA, BRCA1 and the Sonic Hedgehog embryonic development gene 10-kB genomic DNA segment from the functionally extinct Northern White Rhino. Applications of synthetic DNA are then explored through the import of orthogonal transcription and translation molecular machinery into cells, where they directly regulate protein production. On the transcription level, variations in inducible split-T7 polymerase systems are used to create an orthogonal signaling pathway for low leak and tunable transcriptional control of target genes. Unnatural amino acid incorporation is used to translationally regulate genomically modified essential genes, where exposure to this molecule is demonstrated to enable translation of select essential genes. Through the lens of the Central Dogma, in this thesis I will explore various frameworks to build and fine tune the cells we may aspire to create.