Essential (and Dispensable) Genes in Escherichia coli

人们对大肠杆菌K12及其新陈代谢的了解比任何其他自由生活的有机体都多。尽管如此，其38%的基因产物的作用尚不清楚，更不用说这些产物(甚至许多已知的产物)在标准的实验室生长条件下是否具有必要的功能。本研究项目正在使用一种独特的Tn5转座系统来定义大肠杆菌中的必需基因。作为一个重要的副产品，可有可无的基因也将被确定。该项目将使用脉冲转座突变来创建一个转座子标记的插入片段的“完整”文库，将产生从插入的Tn5延伸到相邻序列的转录本，然后将这些转录本与所有4288个开放阅读框(ORF)进行核酸杂交，以跟踪在特定条件下文库所有成员的生长(或未能生长)。这一程序应该允许在确定的生长条件下确定在大肠杆菌K12染色体上的每个已知4288个ORF中的零突变体的生长潜力。此外，如果“完整”文库可重复地缺乏某些代表性，则将调查Tn5转座的体内偏向。虽然对促进对E.ColiK12基因组功能的基本了解有相当大的兴趣，但应该使用的程序也应该有一些重要的应用用途。例如，在开发这一项目时，已经发现了一种技术，该技术应该能够在许多类型的细菌中有效地产生Tn5转座事件，而不需要为各种细菌开发包含Tn5的复制子或在各种细菌中表达Tn5转座酶。这将有助于一些有趣的细菌的转座子突变，传统的遗传方法对这些细菌无效。

More is known about the bacterium Escherichia coli K12 and its metabolism than any other free-living organism. Nonetheless, the role of 38% of its gene products is unknown, much less whether these products (or even many of the known products) are essential functions under standard laboratory growth conditions. This research project is using a unique Tn5 transposition system to define essential genes in E. coli. As an important side product, genes that are dispensable will also be determined. The project will use pulse transposition mutagenesis to create a "complete" library of transposon tagged inserts, transcripts will be generated that extend off of the inserted Tn5s into adjacent sequences and then nucleic acid hybridization of these transcripts to all 4288 open reading frames (ORFs) will be used to follow the growth (or failure to grow) of all members of the library under defined conditions. This procedure should allow the determination of the growth potential of null mutants in each of the known 4288 ORFs in the E. coli K12 chromosome under defined growth conditions. In addition, if the "complete" library reproducibly lacks certain representatives, the in vivo biases of Tn5 transposition will be investigated. While of considerable interest in furthering the basic understanding of E. coli K12 genome function, the procedures that shall be used should also have a number of important applied uses. For instance, in developing this project a technique has been discovered that should be able to efficiently generate Tn5 transposition events in many types of bacteria without the necessity of developing a Tn5 containing replicon for, or expression of the Tn5 transposase in, the various bacteria. This will aid in transposon mutagenesis of a number of interesting bacteria for which conventional genetic approaches have failed.