Brahami Anissa, Castonguay Annie, Déziel Éric
INRS-Institut Armand-Frappier, Laval, QC H7V 1B7, Canada.
Methods Protoc. 2019 Jan 7;2(1):4. doi: 10.3390/mps2010004.
Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen metagenomic libraries for their antibacterial activity against , a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5-10 times more clones per agar plate. Using the E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.
宏基因组技术，尤其是将环境DNA（eDNA）克隆到替代宿主中，使人们能够获取未培养细菌的基因组。然而，仅基于DNA序列确定基因功能仍然是一项重大挑战。宏基因组文库的功能筛选是发现微生物代谢产物的一种有趣方法。我们在此描述一种优化的筛选方法，有助于在大型宏基因组文库中鉴定新型抗菌剂。值得注意的是，我们报告了一种使用DH10B作为替代宿主的详细基因组文库构建方案，并展示了载体/基因组DNA去磷酸化、连接酶失活、连接产物透析以及载体/基因组DNA比例如何极大地影响克隆回收率。此外，我们描述了使用喷枪设备筛选宏基因组文库对金黄色葡萄球菌的抗菌活性，我们将这种方法称为细菌喷雾法。这种细菌喷雾工具极大地促进并改进了大型基因组文库的功能筛选，因为与常用的覆盖法相比，它能够方便地产生更薄且更均匀的目标细菌层，从而使每个琼脂平板上筛选的克隆数量增加5至10倍。以E264基因组DNA作为概念验证，在70000个克隆中有4个抑制了金黄色葡萄球菌的生长，并且发现每个克隆都含有一个DNA插入片段。对这些染色体片段的分析揭示了以前从未报道过负责抗菌剂生产的基因组区域，也未被生物信息学工具预测到。
