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

描述(申请人提供)：本提案回应了广泛的挑战领域“使能技术”，并特别针对06-GM-102*化学家/生物学家合作，通过化学家(S)和生物学家(S)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同基因细菌群体在细胞间转录的差异。这种差异可能是由于双稳定性，这是一种可逆的基因调控表观遗传机制，导致两个群体的出现，一个群体表达一组基因，另一个群体中相同的基因转录沉默。尽管双稳性似乎是微生物应对动态环境的一种非常普遍和高度进化的适应性策略，但目前还没有办法以公正的方式调查整个基因组，寻找对各种环境条件表现出双稳性的基因。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，以表征细菌基因组中每个基因在单个细胞中的表达行为，以响应一系列生物相关条件。为了实现这一目标，我们将采用一种新颖的策略，即先进行一次基因筛选，然后使用微流控平台进行第二次筛选。我们将测试这项技术的功能，确定它是否可以检测到以前发现的霍乱弧菌基因，这些基因的表达受到双稳态控制。将创建一个霍乱弧菌文库，该文库由所有可能的基因与不稳定的绿色荧光蛋白(GFP)融合的细胞组成。这个文库的条目将使用一种新型的微流控平台进行筛选，该平台以高通量一次一个地询问细胞的基因表达。这种方法是史无前例的，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，以及可能发展一个新的科学产业，对经济产生积极影响。 与公共卫生相关：在抗生素治疗期间，少量细菌持续存在的能力阻碍了传染病的控制。这种坚持治疗的人群需要使用长疗程的治疗，并导致治疗失败。该项目将通过开发一种方法和工具来满足这一公共卫生需求，该方法和工具将确定一小部分经过治疗的细菌群体在治疗期间如何变得耐药。这一认识将导致新药和治疗策略的发展。

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.