Development of Universal Genetic Systems for Bacteria

描述（由申请人提供）：数百种细菌的基因组已经被测序，更多的还在后面。然而，可用于基因组操作后测序的通用遗传工具远远缺乏。提出的研究是解决其中一些问题的一个开端。有了这些可用的细菌基因组和不断出现的新出现和再出现的感染性细菌，现有的通用遗传工具应该被探索和进一步修改，以增加这些细菌的有用范围。现有的通用遗传工具将允许对对公共卫生构成严重负担的各种细菌病原体进行研究。因此，我们建议对现有的两种通用工具进行重大改进，即细菌基于rp4的偶联的混杂遗传转移能力和广泛应用的酵母2微米质粒的flip - frt。更具体地说，我们将设计几个基于rp4的结合后自杀捐赠者。这将减轻事先确定适当的选择性媒介的需要，从而扩大这一普遍系统的接收范围。此外，这些工具将在高通量基因组实验中非常有用，补充其他高通量技术，如微阵列，基因组学和蛋白质组学。第二个目标是设计许多有用的结构，包含酵母2微米质粒的独特FRTs（翻转重组靶），用于细菌基因组操作。开发通用的遗传工具来研究细菌是我们的长期目标，因为这将有助于我们实验室和其他实验室的各个研究方面，处理细菌传染病。

DESCRIPTION (provided by applicant): The genomes of hundreds of bacteria have been sequenced, with even more to come. Yet, versatile genetic tools available for genome manipulations post-sequencing are far lacking behind. The proposed research is an initiation to solve some of these problems. With these available bacterial genomes and relentless occurrences of new emerging and reemerging infectious bacteria, existing universal genetic tools should be explored and modified further to increase the range of usefulness in these bacteria. The available universal genetic tools will allow studies to be performed on various bacterial pathogens that are serious burdens to public health. Thus, we are proposing to make significant improvements to two existing universal tools, namely, the promiscuous genetic transfer ability of bacterial RP4-based conjugation and the broadly applied Flp-FRT of the yeast 2-micron plasmid. More specifically, we will engineer several RP4-based conjugal donors that are suicidal post-conjugation. This will broaden the recipient-range of this universal system by alleviating the need for prior determination of appropriate selective media. In addition, such tools will be extremely useful in high-throughput genomic experiments, complementing other high-throughput technologies such as microarrays, genomic, and proteomics. The second aim is to engineer numerous useful constructs, containing unique FRTs (flip-recombination-target) of the yeast 2-micron plasmid, for bacterial genome manipulations. Developing universal genetic tools to study bacteria is our long-term goal, ', since this will aid in various research aspects of our laboratory and others, dealing with infectious diseases of bacteria.