Three major insights: 1) CAST-based barcoding and gene disruption is straightforward, but requires screening and groundwork for quantitative use. 2) The Tn7 insertion locus, when it exists, needs critical evaluation. 3) “tagIMseq” is a versatile, rapid, and easy transposon mapping method

We confirmed rpoZ 3’ UTR insertion had no fitness penalty on plants, and targeted rpoZ in an uncharacterized bacterial pool with a triple tandem guide. After screening colonies (tagIMseq), we subcultured and sequenced genomes from positives, adding perfectly-tagged novel strains to our collection.

We targeted the rpoZ 3’ UTR in a Sphingomonad. We developed a rapid transposon mapping method, “tagIMseq”, leveraging tagmentation and @plasmidsaurus.bsky.social. It even works without a DNA prep on a resuspended colony! Most colonies were good, but many were not, so screening was important.

CASTs can target almost anywhere, so we searched for improved conserved “safe” sites as Tn7 alternatives. Regions between convergently terminating genes downstream of rpoZ in Sphingomonas, or pyrD in Pseudomonas, seemed much better. Both had conserved regions for efficient guides.

But we noticed something uncomfortable about the widely-used “safe” Tn7 site in Sphingomonas. In the majority of cases, insertions there would probably disrupt the next gene, because it was either too close or co-directional. We noticed the same situation in Pseudomonas.

The system let us detect and quantify our tagged strains simultaneously with the uncharacterized bacterial background in a complex host-associated environment.

Amplicon seq quantitatively captured the barcodes (colors) and the native 16S (grey) of the strains. Fascinating that each barcode had a different amplification efficiency (a phenomenon previously reported), requiring empirically-determined correction factors.

We designed our RNA guides within conserved areas so each could be recycled for use on many different strains. We managed insertions in diverse strains, and we could even hit the right target with 5 mismatches in the guide!

In new work led by @ezgiboz.bsky.social, we adapted a single-vector CAST, and made a tag featuring a random DNA barcode between unique and 16S priming sites. We planned “neutral” tags at the Tn7 site downstream of glmS, and “disruptive” tags in a carotenoid gene. www.biorxiv.org/content/10.6...

A quick thread summarizing our new bioRxiv preprint on using CASTs for DNA barcoding to tell "look-alike" bacteria apart (Sphingomonas edition)!! Enjoy the read! ✨

📣Months have raced by, and this year’s committee is already hard at work bringing EEBST 2026 to life! Our abstract submission system is now officially open!! 🚀Deadline April 15th⏰ Join us in sunny Mersin for this exciting event by submitting your latest research at eebst.ekoevo.org Please share!

So happy to be part of the latest issue of New Phytologist! 🌿 Thanks for the kind feature!

Excited to be part of this #NewPhytologistNow webinar on Nov 4! 🌱

Thanks to everyone who came by my poster today for discussion! #2025ISMPMI If you weren’t able to make it but still interested in the potential role native plasmids play within Sphingomonas-plant interactions. Can check it out now on Zenodo zenodo.org/records/1591...

In the greenhouse, #Arabidopsis with hyperactive ACD6 have stronger pathogen defenses but are smaller & make fewer seeds. @derekseveri.bsky.social @plantevolution.bsky.social &co show that in the open field both advantages & disadvantages of the allele disappear @plosbiology.org 🧪 plos.io/465XPDR

This work started nearly 10 years ago and was once my main postdoctoral project at @plantevolution.bsky.social before I slowed work on it to a trickle because it became confusing. But it always remained extremely interesting. journals.plos.org/plosbiology/...

Last week, I had a great time at the New Phytologist EiC Symposium: “Microbes as hidden or prominent players in plant life” in Tartu, Estonia! Honored and very happy to receive the Best Poster Award! ✨ Big thanks to the organizers and all the inspiring scientists I met! 🌱 @newphyt.bsky.social