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This is a very good paper, the authors are explaining the use of BioBloom as a retron based method for barcoded saturation mutagenesis at the scale of a whole bacterial genome, in this case the E. coli genome. They prepared 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. The authors also did a comparison of BioBloom, relative to other techniques like CRISPR enabled mutagenesis or Adaptive Laboratory Evolution, and said it excels at identifying diverse causal SNPs quickly and at smaller working volumes. Moreover, BioBloom makes barcoded saturation mutagenesis accessible at whole genome scale, creating new opportunities for large scale data collection and bacterial engineering. The authors also stated other advantages of using BioBloom over other existing methods, together with its limitations.

Very informative and attractive figures have been used to show and explain: retron recombineering and creation of BioBloom libraries; analysis of library saturation; BioBloom applied to antibiotic resistance and BioBloom experiments investigating salt tolerance. With the latter, also figures showing the genome wide results of salt tolerance experiments and the characterization of salt tolerance variants were present. This research contributes tremendously to the field of bacterial engineering and biotechnology as real -world applications are demonstrated revealing new biology such as the previously unknown salt-tolerance mutations.

Major issue

• The paper is well written; these are some issues when addressed could improve the quality of the paper:

  • The order in which the research sections have been done and presented in the paper is: Abstract, Introduction, Results, Discussion, Methods then Acknowledgments and References. This makes the reader have to flip through different sections before grasping the chronological order of the research.

  • Having a Conclusion section.

  • Referencing style used should be improved so that it is consistent. Also, the in-text citation and the References list should correlate.

Minor issues

• The document is well-written and professionally structured and the following bullets below could improve the overall flow of the research:

  • The addition of 5- 6 keywords after the abstract.

  • If the authors re-organised the sections to be: Abstract, Introduction, Methods, Results, Discussion, Conclusion, Acknowledgements then References.

  • There are some grammatical errors and minor stylistic inconsistencies such as missing spaces after periods, sporadic use of commas, inconsistent formatting for section heads, and occasional run-on sentences. Examples include: "Here. we introduce BioBloom" should not have a period after 'Here'. Phrases like "l ml" should use "1 ml", and "lCso" should be "IC50."

• Overall, these minor issues do not obscure understanding or hinder the logical flow of the document.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.