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测序，还有更多的细菌即将测序。然而，可用于测序后基因组操作的通用遗传工具远远缺乏。拟议的研究是解决这些问题的一个开端。有了这些可用的细菌基因组和不断出现的新出现和重新出现的感染性细菌，应进一步探索和修改现有的通用遗传工具，以增加这些细菌的有用范围。现有的通用遗传工具将使人们能够对严重影响公共卫生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RP 4结合的混杂遗传转移能力和酵母2微米质粒的广泛应用的Flp-FRT。更具体地说，我们将设计几个基于RP 4的结合供体，这些供体在结合后具有自杀性。这将通过减轻预先确定适当的选择性介质的需要来扩大该通用系统的检测范围。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。第二个目标是工程化许多有用的构建体，其含有酵母2微米质粒的独特FRT（翻转重组靶），用于细菌基因组操作。开发研究细菌的通用遗传工具是我们的长期目标，因为这将有助于我们实验室和其他人处理细菌传染病的各个研究方面。

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.