MANIPULATING BACTERIAL GROWTH FOR SINGLE CELL GENOMICS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 流式细胞术和多重置换扩增（MDA）是单细胞基因组学研究的重要手段。 当流式细胞仪从环境中检测和分类细胞候选者时，MDA从微量的DNA模板中随机扩增分离细胞基因组的片段。然后对该扩增过程的产物进行测序和组装以确定分离的细胞的完整基因组身份。由于FACS和MDA是单细胞基因组学的关键工具，因此某些问题是明显的，其导致基因组组装的低效率、高成本、高劳动力过程。在流式细胞仪中，活细胞与来自给定环境样品的颗粒的分化对于确定下游基因组研究的可能细胞候选物至关重要。MDA具有基于基因组模板的GC含量的扩增偏倚。此外，基因组序列中的缺口需要额外的模板来完成序列缺口。如果基因组模板稀缺和/或分离的细胞不能从其环境中回收，则细胞的基因组可能不完整。为了解决这些问题，操纵细菌代表大肠杆菌K12和枯草芽孢杆菌的正常生长周期，以产生大于正常细胞大小和多倍性的表型。一个较大的细胞意味着代谢活动，因此是一个活细胞。如果这样的较大细胞具有其基因组的多个拷贝，则MDA和随后的基因组组装过程可以被简化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. FACS and multiple displacement amplification (MDA) are essential means in the study of single cell genomics. Where FACS detects and sorts cellular candidates from the environment, MDA randomly amplifies segments of the isolated cell's genome from minute quantities of DNA template. The products of this amplification process are then sequenced and assembled to determine the complete genomic identity of the isolated cell. As FACS and MDA are critical tools for single cell genomics, certain issues are evident that contribute to a low- efficient, high-cost, high-labor process of genomic assembly. In FACS, the differentiation of living cells from particles from a given environmental sample is vital to determine possible cellular candidates for downstream genomic studies. MDA has amplification biases based on the genomic template's GC content. Furthermore, gaps in the genomic sequence necessitates additional template to complete the sequence gaps. If the genomic template is scarce and/or the isolated cell cannot be recovered from its environment, then the cell's genome may not be completed. To address these issues, normal growth cycles of bacterial representatives Escherichia coli K12 and Bacillus subtilis were manipulated to result in phenotypes of larger-than-normal cell size and polyploidy. A larger cell suggests metabolic activity and, thus, a living cell. If such a larger cell possesses multiple copies of its genome, then the MDA and subsequent genomic assembly processes may be streamlined.