Modularization and minimization of the yeast transcription factor repertoire

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Transcription factors are proteins that control when a gene gets turned on or off and are essential for controlling the various functions of cells. Here, transcription factors will be organized into modules that turn on particular types of genes and therefore allow researchers to program cells or reprogram cells with the desired functions or applications. Indeed, the ability to program or reprogram cells on demand has long been a goal of synthetic biology and bioengineering, as this ability would dramatically impact medicine and the development of biotechnology. Training of diverse early career scientists will play a central role in the proposed project, providing post-doctoral researchers, Ph.D. students and undergraduates with valuable expertise in an area of rapidly growing importance to the bioeconomy. A S. cerevisiae transcription factor module will be built as a neochromosome containing ~200 validated and putative DNA-binding, RNA polymerase II-associated transcription factors. Transcription factor-free versions of the 16 native yeast chromosomes will be constructed through the cloning and reassembly of transcription factor-free chromosomal segments. This clone-and-reassemble strategy solves a persistent challenge in genetics and genomics, namely the precise and simultaneous deletion of large numbers of genes. This part of the project will produce a unique and powerful platform in which all transcription factor activity arises from the neochromosome. Taking advantage of this novel platform, the transcription factor module will be reduced to a minimal set sufficient for robust yeast viability. This minimization will be done using an experimental strategy for introducing large numbers of random transposon insertions into targeted specific regions of the genome. This research will empirically answer questions about the numbers and identities of the minimal sets of TFs that can maintain yeast viability both generally and in specific conditions. This part of the project will culminate in the synthesis of a transcription factor module containing only a minimal set of transcription factors. This project will be integrated into a capstone course for undergraduates interested in biotechnology. The course’s curriculum will include a combination of lectures and hands-on activities. Students will be exposed to both the computational and wet lab components of synthetic genomics. The students will learn to design synthetic DNA, assemble gene-sized molecules into larger constructs comprised of multiple genes, verify these assemblies by sequencing, analyze the data, and combine these larger constructs into chromosome-sized molecules in yeast.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.