Essential (and Dispensable) Genes in Escherichia coli

点击翻译按钮获取中文摘要

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.