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模块，这些模块启动特定类型的基因，从而允许研究人员对细胞编程或重新编程细胞，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新编程的能力长期以来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。对不同的早期职业科学家的培训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日益重要的领域的宝贵专业知识。一个酿酒酵母转录因子模块将被构建为一个新染色体，其中包含大约200个有效的和假定的DNA结合、RNA聚合酶II相关的转录因子。通过克隆和重组无转录因子的染色体片段，将构建16条天然酵母染色体的无转录因子版本。这种克隆和重组策略解决了遗传学和基因组学中的一个长期挑战，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，在这个平台上，所有转录因子的活性都来自新染色体。利用这个新的平台，转录因子模块将被减少到足以保证酵母活力的最小集合。这种最小化将使用一种实验策略来实现，该策略将大量随机转座子插入到基因组的特定目标区域。这项研究将经验性地回答有关能够在一般和特定条件下维持酵母活性的最小转铁蛋白集合的数量和身份的问题。该项目的这一部分将最终合成一个只包含最小转录因子集的转录因子模块。该项目将被整合为对生物技术感兴趣的本科生的顶峰课程。这门课程的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部分。学生们将学习设计合成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，并在酵母中将这些更大的结构组合成染色体大小的分子。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.