Modularization and minimization of the yeast transcription factor repertoire

转录因子是控制基因何时开启或关闭的蛋白质，对控制细胞的各种功能至关重要。在这里，转录因子将被组织成打开特定类型基因的模块，从而允许研究人员对细胞进行编程或重新编程，使其具有所需的功能或应用。事实上，根据需要对细胞进行编程或重编程的能力一直是合成生物学和生物工程的目标，因为这种能力将极大地影响医学和生物技术的发展。培养不同的早期职业科学家将在拟议的项目中发挥核心作用，为博士后研究人员、博士生和本科生提供对生物经济迅速增长的重要领域的宝贵专业知识。将构建酿酒酵母转录因子模块，作为包含约200个已验证和推测的dna结合，RNA聚合酶ii相关转录因子的新染色体。通过对无转录因子染色体片段的克隆和重组，构建16条原生酵母染色体的无转录因子版本。这种克隆重组策略解决了遗传学和基因组学中一个持续存在的挑战，即大量基因的精确和同时缺失。该项目的这一部分将产生一个独特而强大的平台，其中所有转录因子活性都来自新染色体。利用这种新颖的平台，转录因子模块将减少到一个最小的集合，足以稳健的酵母活力。这种最小化将通过将大量随机转座子插入到基因组的特定区域的实验策略来实现。这项研究将根据经验回答有关在一般和特定条件下维持酵母活力的最小TFs集的数量和身份的问题。该项目的这一部分将最终合成一个转录因子模块，其中只包含一组最小的转录因子。这个项目将被整合到对生物技术感兴趣的本科生的一门顶点课程中。课程将包括讲座和实践活动的结合。学生将接触到合成基因组学的计算和湿实验室组件。学生将学习设计合成DNA，将基因大小的分子组装成由多个基因组成的更大的结构，通过测序验证这些组装，分析数据，并将这些更大的结构组合成酵母中的染色体大小的分子。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.