Measuring whole genome expression, cell by cell: bistability in Vibrio cholerae

描述（由申请人提供）：本提案响应了广泛的挑战领域“使能技术”，具体针对06-GM-102* 化学家/生物学家合作，通过化学家和生物学家之间的合作促进工具开发。单细胞技术的最新进展揭示了生活在相同同质环境中的细菌的同基因群体中的细胞间转录差异。这种差异可能是由于双稳态，一种可逆的基因调控表观遗传机制，导致两个群体的出现，一个部分表达一组基因，另一个部分中相同的基因是转录沉默的。虽然双稳态似乎是一种非常普遍和高度进化的适应性策略，微生物通过这种策略与动态环境相抗衡，但目前还没有办法以公正的方式调查整个基因组，以寻找在各种环境条件下表现出双稳态的基因。在这个为期两年的项目中，一个由化学家和生物学家组成的团队将开发一种强大且广泛适用的方法，以表征单个细胞在一系列生物相关条件下细菌基因组中每个基因的表达行为。为了实现这一目标，我们将采用一种新的策略，将初级遗传筛选与使用微流体平台的第二次筛选相结合。我们将测试这项技术的功能，确定它是否可以检测先前确定的霍乱弧菌基因的表达是在细菌控制下。将创建由具有融合到不稳定的绿色荧光蛋白（GFP）作为报告基因的所有可能基因的细胞组成的霍乱弧菌文库。将使用一种新的微流体平台来筛选该文库的扩增产物，该微流体平台以高通量一次一个地询问细胞的基因表达。这种方法是前所未有的，这种工具一旦完善，应有助于更好地了解生态和控制传染病。目前的提案将通过在斯坦福大学创造或保留七个就业机会以及通过可能发展一个新的科学产业来对经济产生积极影响。 公共卫生相关性：在抗生素治疗期间，少量细菌能够持续存在，这阻碍了传染病的控制。这种持续性人群是需要使用长疗程治疗和治疗失败的原因。该项目将通过开发一种方法和工具来解决这一公共卫生需求，该方法和工具将确定一部分经过治疗的细菌群体如何在治疗期间变得耐药。这种理解将导致新药物和治疗策略的开发。

DESCRIPTION (provided by applicant): This proposal is responsive to the broad Challenge Area "Enabling Technologies" and specifically is directed to 06-GM-102* Chemist/biologist collaborations facilitating tool development through collaborations between a chemist(s) and a biologist(s). Recent advances in single-cell technology have revealed cell-to-cell transcriptional differences in isogenic populations of bacteria living in the same homogeneous environment. Differences of this kind can be due to bistability, a reversible epigenetic mechanism of gene regulation that causes the appearance of two populations, one fraction that expresses a set of genes and the other in which the same genes are transcriptionally silent. Though bistability appears to be a very general and highly evolved adaptive strategy by which microbes contend with dynamic environments, there is currently no way to survey an entire genome in an unbiased manner for genes that exhibit bistability in response to a variety of environmental conditions. During this two year project, a team of chemists and biologists will develop a robust and broadly applicable method to characterize the expression behavior of each gene in a bacterial genome, by individual cells, in response to a suite of biologically relevant conditions. To achieve this, we will pursue a novel strategy that combines a primary genetic screen followed by a second screen using a microfluidic platform. We will test the functionality of this technology by determining if it can detect previously identified V. cholerae genes whose expression is under bistable control. A V. cholerae library will be created composed of cells with all possible genes fused to unstable green fluorescent protein (GFP) as a reporter. Entries of this library will be screened using a novel microfluidic platform that interrogates cells for gene expression, one at a time, with a high throughput. This approach is unprecedented and such a tool, when perfected, should contribute to an improved understanding of the ecology and control of infectious diseases. The current proposal would positively impact the economy by creating or retaining seven jobs at Stanford University and through the possible development of a new scientific industry. PUBLIC HEALTH RELEVANCE: The control of infectious diseases is impeded by the capacity of small numbers of bacteria to persist during antibiotic treatment. This persistor population is responsible for the need to use long courses of therapy and for therapeutic failures. This project will address this public health need by developing a method and tool that will identify how a fraction of a treated population of bacteria becomes drug tolerant during treatment. This understanding will lead to the development of new drugs and treatment strategies.