Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800, Kongens Lyngby, Denmark.
Department of Systems Biology, Columbia University, New York, NY, USA.
Sci Rep. 2018 May 3;8(1):6961. doi: 10.1038/s41598-018-25201-5.
Adaptive laboratory evolution is an important tool to evolve organisms to increased tolerance towards different physical and chemical stress. It is applied to study the evolution of antibiotic resistance as well as genetic mechanisms underlying improvements in production strains. Adaptive evolution experiments can be automated in a high-throughput fashion. However, the characterization of the resulting lineages can become a time consuming task, when the performance of each lineage is evaluated individually. Here, we present a novel method for the markerless insertion of randomized genetic barcodes into the genome of Escherichia coli using a novel dual-auxotrophic selection approach. The barcoded E. coli library allows multiplexed phenotyping of evolved strains in pooled competition experiments. We use the barcoded library in an adaptive evolution experiment; evolving resistance towards three common antibiotics. Comparing this multiplexed phenotyping with conventional susceptibility testing and growth-rate measurements we can show a significant positive correlation between the two approaches. Use of barcoded bacterial strain libraries for individual adaptive evolution experiments drastically reduces the workload of characterizing the resulting phenotypes and enables prioritization of lineages for in-depth characterization. In addition, barcoded clones open up new ways to profile community dynamics or to track lineages in vivo or situ.
适应性实验室进化是一种将生物体进化为对不同物理和化学胁迫的耐受性增加的重要工具。它被应用于研究抗生素抗性的进化以及生产菌株改进的遗传机制。适应性进化实验可以以高通量的方式自动化。然而，当逐个评估每个谱系的性能时，对产生的谱系进行表征可能会成为一项耗时的任务。在这里，我们提出了一种使用新型双重营养缺陷型选择方法将随机遗传条形码插入大肠杆菌基因组中的无标记插入方法。带有条形码的大肠杆菌文库允许在混合竞争实验中对进化的菌株进行多重表型分析。我们在适应性进化实验中使用了这个带有条形码的文库，以进化对三种常见抗生素的抗性。通过将这种多重表型分析与传统的药敏试验和生长速率测量进行比较，我们可以证明这两种方法之间存在显著的正相关。使用带有条形码的细菌菌株文库进行个体适应性进化实验可以大大减少对产生的表型进行特征描述的工作量，并能够对进行深入特征描述的谱系进行优先级排序。此外，条形码克隆为分析群落动态或在体内或原位跟踪谱系开辟了新途径。
