用于功能宏基因组学的广宿主范围克隆

Wide host-range cloning for functional metagenomics.

作者信息

Wexler Margaret, Johnston Andrew W B

机构信息

School of Biological Sciences, University of East Anglia, Norwich, Norfolk, UK.

出版信息

Methods Mol Biol. 2010;668:77-96. doi: 10.1007/978-1-60761-823-2_5.

DOI:10.1007/978-1-60761-823-2_5

PMID:20830557

Abstract

We describe how wide host-range cloning vectors can lead to more flexible and effective procedures to isolate novel genes by screening metagenomic libraries in a range of bacterial hosts, not just the conventionally used Escherichia coli. We give examples of various wide host-range plasmid, cosmid, and BAC cloning vectors and the types of genes and activities that have been successfully obtained to date. We present a detailed protocol that involves the construction and screening of a metagenomic library comprising fragments of bacterial DNA, obtained from a wastewater treatment plant and cloned in a wide host-range cosmid. We also consider future prospects and how techniques and tools can be improved.

摘要

我们描述了广宿主范围克隆载体如何能够带来更灵活、有效的程序，通过在一系列细菌宿主（而不仅仅是传统使用的大肠杆菌）中筛选宏基因组文库来分离新基因。我们给出了各种广宿主范围质粒、黏粒和细菌人工染色体（BAC）克隆载体的例子，以及迄今已成功获得的基因类型和活性。我们提出了一个详细的方案，该方案涉及构建和筛选一个宏基因组文库，该文库包含从污水处理厂获得并克隆到广宿主范围黏粒中的细菌DNA片段。我们还考虑了未来的前景以及如何改进技术和工具。