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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

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Reviewer #1: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

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Reviewer #1: The manuscript by Sawitzke et al entitled “Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation” shows how a deletion of the xonA gene, which encodes a 3’ ssDNA exonuclease from E. coli, greatly stimulates the in vivo cloning capabilities following electroporation of linear DNA fragments into a strain that expresses the RecET phage recombination system. The authors first show what was already known, that the RecET system is more efficient at linear DNA fragment annealing compared to the lambda Red recombination system. They test increasing homology lengths where they show that the higher efficiency of the RecET system is most pronounced as smaller homologies (~50 bp).

Their most significant finding is the deletion of xonA results in higher efficiencies with both systems, with up to a 70-fold increase in the case of RecET acting on 2 or 3 overlapping linear fragments. Significantly, the RecET-xonA system allowed 50 bp homologies to work efficiently, allowing homologies to be added to the primers during PCR-amplification of linear DNA fragment substrates. Also, the RecET-xonA system showed the greatest effect relative to the xonA+ strain, where the recombination efficiency was increased 4 orders of magnitude when six fragments with overlapping homologies were used to assemble the recombinant plasmid. In all cases, they followed correct plasmid assembly by selecting of drug-resistant markers or the presence of blue colonies with annealed fragments due to the presence of the lacZ gene in the recombinant plasmid; the accuracy was generally 90% or greater.

Finally, the authors finally compare their in vivo system to NEB’s Gibson assembly in vitro kit and find the two systems generally compatible, suggesting that use of the RecET-xonA strain would save time without the need to treat the linear fragments with DNA modifying enzymes in vitro.

The manuscript is well written and the figures are clear. The experiments are sound and appropriate controls and statistical analyses are appropriate.

Minor points:

The only difficulty I found was that the Figures were not labelled. In particular, the last figure in manuscript entitled “DNA Fragment Assembly” is not discussed in the text. Should this figure be labelled Figure 7B?

On page 6, the reference to “Figure S3” is not correct. Fig. S3 contains lacZ sequence information from white colonies, not patching colonies on to LB-kan or LB-cam plates. Is there some other Supp Figure that shows the patching results?

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Reviewer #1: No

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Enhancement of RecET-mediated in vivo linear DNA assembly by a xonA mutation

PONE-D-25-63366R1

Dear Dr. Sawitzke,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Bashir Sajo Mienda, PhD

Academic Editor

PLOS One

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