Commensal bacteria from the human intestinal microbiota play important roles in health and disease. Research into the mechanisms by which these bacteria exert their effects is hampered by the complexity of the microbiota, the strict growth requirements of the individual species and a lack of genetic tools and resources. The assembly of ordered transposon insertion libraries, in which nearly all nonessential genes have been disrupted and the strains stored as independent monocultures, would be a transformative resource for research into many microbiota members. However, assembly of these libraries must be fast and inexpensive in order to empower investigation of the large number of species that typically compose gut communities. The methods used to generate ordered libraries must also be adapted to the anaerobic growth requirements of most intestinal bacteria. We have developed a protocol to assemble ordered libraries of transposon insertion mutants that is fast, cheap and effective for even strict anaerobes. The protocol differs from currently available methods by making use of cell sorting to order the library and barcoded transposons to facilitate the localization of ordered mutations in the library. By tracking transposon insertions using barcode sequencing, our approach increases the accuracy and reduces the time and effort required to locate mutants in the library. Ordered libraries can be sorted and characterized over the course of 2 weeks using this approach. We expect this protocol will lower the barrier to generating comprehensive, ordered mutant libraries for many species in the human microbiota, allowing for new investigations into genotype–phenotype relationships within this important microbial ecosystem.

来自人类肠道微生物群的共生细菌在健康和疾病中发挥着重要作用。由于微生物群的复杂性、个体物种的严格生长要求以及缺乏遗传工具和资源，对这些细菌发挥作用的机制的研究受到了阻碍。有序转座子插入文库的组装，其中几乎所有非必需基因都已被破坏，菌株作为独立的单一培养物储存，将成为研究许多微生物群成员的变革性资源。然而，这些文库的组装必须快速且成本低廉，以便能够对通常构成肠道群落的大量物种进行调查。用于生成有序库的方法还必须适应大多数肠道细菌的厌氧生长要求。我们开发了一种协议来组装有序的转座子插入突变体库，该库快速、便宜且对甚至严格的厌氧菌也有效。该协议与当前可用的方法不同，它利用细胞分类来订购库和条形码转座子，以促进库中有序突变的本地化。通过使用条形码测序跟踪转座子插入，我们的方法提高了准确性并减少了在库中定位突变体所需的时间和精力。使用这种方法，可以在 2 周内对有序库进行排序和表征。我们预计该协议将降低生成综合性的障碍，