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Exploitation of new bacteriophages for generic strain engineering methods and functional genomic analysis of diverse bacteria

利用新型噬菌体进行通用菌株工程方法和多种细菌的功能基因组分析

基本信息

批准号：
BB/G000298/1
负责人：
George Salmond
金额：
$ 12.84万
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2008
资助国家：
英国
起止时间：
2008 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FG000298%2F1
关键词：
Exploitation new bacteriophages generic strain

项目摘要

Until the advent of rapid gene cloning and high throughput DNA sequencing methods, there was only very limited knowledge on the genetics, biochemistry and physiology of most bacteria. Prior to the development of such technologies, the most sophisticated studies on bacteria were done with E. coli K12 strains for which there was tremendous background information based on the ability to do inventive genetic analysis and strain constructions by a combination of in vivo and in vitro genetic manipulation. In recent years the ability to determine genome sequences has advanced at an incredible pace and so it is now possible to determine the complete DNA sequence of a new bacterium within a day (although gene annotation takes considerably longer). Additional technical advances in the methodologies for studying gene expression (e.g. by Q-RT-PCR analysis) have enhanced significantly the ability to investigate regulation of expression of specific genes in bacteria for which there are no, or minimal, genetic analysis methods available. However, a bottleneck in the full and meaningful exploitation of total genomic sequence information in 'new' bacteria is often the ability to make defined, specific mutants and to genetically complement such mutants for physiologically rigorous studies in transcriptomics, proteomics and metabolomics. To achieve this the researcher usually has to try to transfer existing genetic and molecular biology methods for mutagenesis and complementation (usually plasmid-based) from well-studied bacteria - with extremely variable outcomes. In the absence of rigorously defined mutants and clean complementation strategies, the veracity of comparative 'omic studies is questionable, at best, and non-existent at worst. Consequently it would be very useful to have facile and robust methods for transferring defined mutant genes between strains and for strain engineering in bacteria for which the full genomic sequences are known, but for which there is little other information - except bioinformatic prediction. In this project we will isolate and develop some bacterial viruses - generalised transducing phages (GTPs) - for a range of bacterial hosts which have been genomically sequenced, but for which there is little in the way of genetic engineering methodology currently available. These GTPs will be useful for bacterial strain constructions that are required for robust comparisons of wild type and mutant strains in functional genomics research programmes. In addition, we will start the engineering of a new bacteriophage (phiNP) that we discovered in a bacterial mouse pathogen. This phage is temperate and integrates its genome into the bacterial chromosome in single copy at a precise location towards the end of a bacterial gene involved in the efficient control of protein synthesis and ribosome recycling (tmRNA or ssrA). When the phage genome integrates into the bacterial gene sequence it creates a target sequence duplication such that the tmRNA target gene is functionally reconstituted and thus there is no obvious defect in the bacterial host as a consequence of acquiring the virus DNA. The bacterial tmRNA gene is very widely distributed in bacteria and is even found in plastid genomes of higher cells. Consequently, this bacterial virus could be manipulated to make derivatives that will provide tools for transferring mutant and normal genes, mutagenic transposons and other genetic elements into the chromosomes of bacterial hosts containing the conserved target sequence. Therefore, by gene engineering methods, we intend to derive phiNP-based tools with a broad host range applicability in mutagenesis, cloning and complementation analysis. The combination of GTPs and phiNP-derived technologies will broaden the number of bacterial hosts for which powerful functional genomics can be performed and this should enfranchise more researchers for diverse - and rigorously controlled - 'omic studies.

在快速基因克隆和高通量 DNA 测序方法出现之前，人们对大多数细菌的遗传学、生物化学和生理学的了解非常有限。在开发此类技术之前，对细菌最复杂的研究是用大肠杆菌 K12 菌株进行的，该菌株具有基于通过体内和体外遗传操作相结合进行创造性遗传分析和菌株构建的能力的巨大背景信息。近年来，确定基因组序列的能力以令人难以置信的速度进步，因此现在可以在一天内确定新细菌的完整 DNA 序列（尽管基因注释需要相当长的时间）。研究基因表达的方法学（例如通过 Q-RT-PCR 分析）的其他技术进步显着增强了研究细菌中特定基因表达调控的能力，而对于这些细菌，没有或只有很少的遗传分析方法可用。然而，充分且有意义地利用“新”细菌的总基因组序列信息的瓶颈通常是制造明确的、特定的突变体并在遗传上补充这些突变体以用于转录组学、蛋白质组学和代谢组学的生理学严格研究的能力。为了实现这一目标，研究人员通常必须尝试转移现有的遗传和分子生物学方法，以从经过充分研究的细菌中进行诱变和互补（通常基于质粒），结果差异极大。在缺乏严格定义的突变体和干净的互补策略的情况下，比较组学研究的准确性充其量是值得怀疑的，而最坏的情况则是不存在。因此，拥有简单而稳健的方法来在菌株之间转移确定的突变基因以及在已知完整基因组序列但除了生物信息学预测之外几乎没有其他信息的细菌中进行菌株工程将是非常有用的。在这个项目中，我们将分离和开发一些细菌病毒——通用转导噬菌体（GTP）——针对一系列已进行基因组测序的细菌宿主，但目前可用的基因工程方法很少。这些 GTP 对于功能基因组学研究项目中野生型和突变株的稳健比较所需的细菌菌株构建很有用。此外，我们将开始对我们在细菌性小鼠病原体中发现的新型噬菌体（phiNP）进行工程设计。这种噬菌体是温和的，并将其基因组以单拷贝的形式整合到细菌染色体中，位于细菌基因末端的精确位置，该基因参与有效控制蛋白质合成和核糖体回收（tmRNA 或 ssrA）。当噬菌体基因组整合到细菌基因序列中时，它会产生靶序列重复，从而使 tmRNA 靶基因在功能上重建，因此获得病毒 DNA 后，细菌宿主不会出现明显缺陷。细菌tmRNA基因在细菌中分布非常广泛，甚至在高等细胞的质体基因组中也有发现。因此，可以操纵这种细菌病毒来制造衍生物，这些衍生物将提供将突变和正常基因、诱变转座子和其他遗传元件转移到含有保守靶序列的细菌宿主染色体中的工具。因此，通过基因工程方法，我们打算衍生出基于 phiNP 的工具，在诱变、克隆和互补分析中具有广泛的宿主范围适用性。 GTP 和 phiNP 衍生技术的结合将扩大可以进行强大功能基因组学的细菌宿主的数量，这将使更多研究人员能够进行多样化且严格控制的组学研究。