BioBloom, a method for barcoded saturation mutagenesis of an entire bacterial genome

Schubert, M. G.; Sukarto, E.; Martin, F. R.; Mancuso, J. E.; Spens, A.; Pedersen, T.; Isaev, K.; Greene, H.; Hicks, N. D.; Nattermann, U.; Liu, J.; Stork, D. A.; DeBenedictis, E. A.

doi:10.64898/2026.02.04.703572

bioRxiv · 10.64898/2026.02.04.703572

BioBloom, a method for barcoded saturation mutagenesis of an entire bacterial genome

Schubert, M. G.·Sukarto, E.·Martin, F. R.·Mancuso, J. E.·Spens, A.·Pedersen, T.·Isaev, K.·Greene, H.·Hicks, N. D.·Nattermann, U.·Liu, J.·Stork, D. A.·DeBenedictis, E. A.

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Abstract

Saturation mutagenesis is a powerful tool for understanding and engineering the function of biological systems. and has been applied successfully to characterize the mutational landscape of individual proteins and genetic loci. However. it has not been applied at the whole-genome scale due to the challenges of both creating and quantifying a saturating set of mutations. Here we introduce BioBloom. a retron-based method for barcoded saturation mutagenesis at the scale of a whole bacterial genome. We constructed a barcoded BioBloom library with >99% projected sampling of saturating single - nucleotide polymorphism (SNP) mutations of the E. coli genome, and applied it to identify beneficial mutations under salt and antibiotic selection. Relative to other techniques like CRISPR-enabled mutagenesis or Adaptive Laboratory Evolution, BioBloom excels at identifying diverse causal SNPs quickly and at smaller working volumes. A barcoded, saturating mutation library is also a shared resource, and we are releasing the updated BioBloom-Ec.-2.0 library to the scientific community for broader adoption and application. Together. BioBloom makes barcoded saturation mutagenesis accessible at whole-genome scale, creating new opportunities for large-scale data collection and bacterial engineering.

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Schubert, M. G., Sukarto, E., Martin, F. R., Mancuso, J. E., Spens, A., Pedersen, T., Isaev, K., Greene, H., Hicks, N. D., Nattermann, U., Liu, J., Stork, D. A., DeBenedictis, E. A.. 2026-02-05. BioBloom, a method for barcoded saturation mutagenesis of an entire bacterial genome. https://doi.org/10.64898/2026.02.04.703572

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