Finally, we made “magic pools” of these BarTn7 vectors, which use a parts-based strategy to screen promoters and antibiotic markers in new species. These magic pools identified vector designs which enabled BarTn7 in bacteria which were recalcitrant to the original plasmids!

We then took steps to optimize the system by reversing cargo orientation to prevent host toxicity and combining transposon and transposase into one vector, improving conjugation efficiency by ~10X

BarTn7 was also more accurate than 16S sequencing at measuring community composition when different barcoded species were combined and outgrown in different media, and comparable to Illumina shotgun whole-genome sequencing (WGS) at a fraction of the cost

Next, we show that BarTn7 enables evolution experiments by identifying adaptive lineages (and the rise and fall of rare lineages). We detected selective sweeps linked to causal folA mutations in E. coli and Klebsiella treated with trimethoprim

Then we use BarTn7 to measure the colonization of Switchgrass roots by three plant-associated bacteria and find that lineage recruitment was species-specific: Pseudomonas (WCS417) often outcompeted lineages of the same species, while Paraburkholderia (Bfirm) colonized more evenly

First, we confirm that each lineage grows neutrally, showing population-level changes are due to drift or ecological dynamics rather than barcode bias

By chromosomally integrating DNA barcodes using the Tn7 transposon we created near-isogenic populations tagged with unique barcodes to we can track lineage behavior at the sub-species level