Giuliani Maria, Parrilli Ermenegilda, Pezzella Cinzia, Rippa Valentina, Duilio Angela, Marino Gennaro, Tutino Maria Luisa
Department of Organic Chemistry and Biochemistry, Università degli studi di Napoli Federico II, Naples, Italy.
Methods Mol Biol. 2012;824:219-33. doi: 10.1007/978-1-61779-433-9_11.
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.
对海洋南极嗜盐浮游假交替单胞菌TAC125基因组的测序和注释为研究其适应寒冷条件的分子机制铺平了道路。对这种独特细菌的兴趣日益浓厚，促使人们开发了几种用于在分子水平上研究它的遗传工具。为了更深入地了解PhTAC125的生理学，开发了一种用于该细菌反向遗传学的遗传系统。在本研究中，我们描述了一种通过框内缺失进行等位基因交换和/或基因失活以及在嗜盐浮游假单胞菌中使用反选择标记的实用技术。详细报道了合适的非复制性质粒的构建以及用于在该细菌中进行两步整合-分离策略的方法。此外，还描述了两个实例，其中应用所开发的方法来确定基因功能或构建基因工程细菌菌株。
