A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125
A Novel Strategy for the Construction of Genomic Mutants of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125
复制标题
DOI：
10.1007/978-1-61779-433-9_11
复制
发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Tutino, Maria Luisa
中科院分区：
文献类型：
--
作者：
Giuliani, Maria;Parrilli, Ermenegilda;Tutino, Maria Luisa
海洋南极盐浮游假异单胞菌TAC125基因组的测序和注释为研究其适应寒冷环境的分子机制奠定了基础。人们对这种独特细菌的兴趣日益浓厚，促使人们开发了几种基因工具，用于在分子水平上研究它。为了更深入地了解PhTAC125的生理机制，我们建立了该细菌的反向遗传系统。在目前的工作中，我们描述了一种实用的技术，用于等位基因交换和/或基因失活的框架内缺失和使用一个反选择的标记在P. haloplankton。本文详细报道了该细菌非复制质粒的构建和两步整合分离策略的方法。此外，还描述了两个实例，其中所开发的方法被应用于发现基因功能或构建基因工程菌株。
The sequencing and the annotation of the marine Antarctic Pseudoalteromonas haloplanktis TAC125 genome has paved the way to investigate on the molecular mechanisms involved in adaptation to cold conditions. The growing interest in this unique bacterium prompted the developing of several genetic tools for studying it at the molecular level. To allow a deeper understanding of the PhTAC125 physiology a genetic system for the reverse genetics in this bacterium was developed. In the present work, we describe a practical technique for allelic exchange and/or gene inactivation by in-frame deletion and the use of a counterselectable marker in P. haloplanktis. The construction of suitable non-replicating plasmid and methods used to carry out a two-step integration segregation strategy in this bacterium are reported in detail.Furthermore two examples, in which the developed methodology was applied to find out gene function or to construct genetically engineered bacterial strains, were described.