Abstract
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.
Highlights
Lineage tracking is a valuable tool used to answer many fundamental biological questions regarding the evolutionary forces and principles behind adaptation processes[1]
Especially those that confer antibiotic resistance, could potentially interfere with our interest of studying de novo antibiotic resistance mutations, we developed a protocol that allows the marker-free insertion of genetic barcodes into the genome of E. coli
Barcoded strains are highly useful to study the population dynamics, those that result from random processes e.g. population bottlenecks and genetic drift that are not driven by mutant selection, as well as evolutionary processes of heterogenic populations
Summary
Lineage tracking is a valuable tool used to answer many fundamental biological questions regarding the evolutionary forces and principles behind adaptation processes[1]. Later fluorescent tags were used in microscopy to follow specific phenotypes over time[14,15], during development in vivo[16,17,18], in cancer models[17] or to trace individual lineages of cells in a population[19,20]. Whole genome sequencing enabled researchers to follow the evolution of individual MRSA strains in a human host during antibiotic treatment[21], to characterize the colonialization of the infant gut[10,11] or to perform multiplexed phenotyping of different gut microbiome species[22]. We showed, as a proof of concept that the multiplexed phenotyping enabled through the barcoding scheme presented here, correlates well with traditional phenotyping methods based on individual clones and allows deeper insights into the population dynamics that would not be detected by traditional growth assays
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