A convenient five-segment cassette procedure for DNA insertions coding for novel peptides

Jonathan Filley

doi:10.1371/journal.pone.0307713

Peer Review History

Original SubmissionApril 13, 2024
31 May 2024 Decision Letter - Dhana Govind Gorasia, Editor PONE-D-24-14914A convenient five-segment cassette procedure for DNA insertions coding for novel peptidesPLOS ONE Dear Dr. Filley, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 15 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Also, in the letter to the editor, the authors have mentioned that they are preparing another manuscript with all the results going in that second manuscript. This manuscript can only proceed if the figures requested by reviewer #2 are included in the current manuscript. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 3. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This study describes a method for the convenient access to peptide mutation libraries with particular consideration to the economic assembly of DNA cassettes. The protocols appear to have been well executed, the advantages of the five segment insertion method demonstrated, and the structure/properties of the resultant peptides considered in the discussion. The authors have also appropriately discussed the scope and limitations of the approached described in the text. Reviewer #2: This manuscript describes used of short oligonucleotides to make hybrid cassettes for ligation to the 3’ of a GFP-encoding gene in a plasmid. The effect of various fusions on GFP- peptide fusion product fluorescence, solubility and affinity for nickel-chelate resin is described although no experimental data such as gels showing solubility/insolubility of GFP to support the conclusion is presented. The conclusion is that use of short overlapping oligonucleotides can expedite production of recombinant peptides with targeted mutation and be a cost and time-effective alternative to peptide synthesis or use of longer intact DNAs as plasmid inserts. The paper describes only one protein, GFP as fusion partner with the coded peptides. Thus, it is a study of the effect of changing C-terminal peptide sequences on GFP. The cassette approach is claimed to be cost effective relative to purchase of short peptides, but with a cost of $60 for a peptide (dependent on locale) the cost of buying the oligonucleotides, cloning, harvesting plasmids, sequencing and then examining the protein produced would be similarly expensive and not that much shorter in time frame. The advantage of the method is the fact that the fused protein is large, which would be prohibitive to synthesis. This should be emphasized. Also, the method could viably be used for internal sequence replacement in a gene coding a protein of interest. Abstract: Describes the work adequately. Introduction: Sufficient. Method: I have questions regarding methods. To ensure obtained sequence changes are as expected, do the oligonucleotides used require purification eg by HPLC or just desalting? The author refers to denaturation of the restriction endonuclease. How is this achieved? If by heating, which is the standard practice, how does this affect the insert DNAs? If by addition of chemicals, how is it ensured that ligation is not inhibited? If there is a small change to replace for example a single nucleotide for an amino acid substitution, is it necessary to have the reverse complements strand with this change? I would presume this would give a 50:50 rate of wild type vs mutated sequence in retrieved plasmids. Was this tested? Has the author used degenerate oligonucleotides to produce mutated peptides? Please comment here or in discussion. Results and discussion: Page 13: with respect to the conclusion that “close to eight polar residues are required for good solubility when designing 20 amino acid tails.” Since GFP was the only protein tested as a fusion, the statement should be more conservative and state that “close to eight polar residues are required for good solubility when designing 20 amino acid tails appended to GFP”. Were any analyses performed to determine what proportion of ALL colonies, brightly fluorescent or otherwise, contained plasmids with synthetic DNA inserts? If so, what is the nucleotide sequence error rate for ALL plasmids with inserts? Page 14: “Thus, the five segment insertion technique described here has an error rate estimated to be close to 1%.” Because the error rate for ALL plasmids produce was not reported, this should read “Thus, for recombinants with bright GFP fluorescence the five segment insertion technique described here has an error rate estimated to be close to 1%.” Overall, the results as described (but not presented) show promise for the versatility of the system and proof of principle. However, the versatility for carrier proteins which do not have a clearly discernible phenotype like GFP is unknown. With no indication of the true successful insert retention and so the true percent insert error rate, the usefulness for rapid selection of recombinant fusion genes is also not known. This should be made clear in this short report. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0307713.r001
Revision 1
29 Jun 2024 Author Response Reviewer #2 has raised valid concerns which need to be addressed. Also, in the letter to the editor, the authors have mentioned that they are preparing another manuscript with all the results going in that second manuscript. This manuscript can only proceed if the figures requested by reviewer #2 are included in the current manuscript. Response: The concerns of Reviewer #2 are addressed below. “Another manuscript” mentioned above is not in preparation. The future work referred to in the cover letter is yet to be determined, but will include the protocols used in the current manuscript. Thus, the current manuscript (if accepted) will be cited in the “Experimental” section of the future work. The author was unable to locate in the reviewer’s comments any mention of requested figures by Reviewer #2. Review Comments to the Author (from Reviewer #2) a. The effect of various fusions on GFP- peptide fusion product fluorescence, solubility and affinity for nickel-chelate resin is described although no experimental data such as gels showing solubility/insolubility of GFP to support the conclusion is presented. Response: The criterion for solubility of the fusions is the observation of green fluorescence from either the supernatant (soluble) or the pellet (insoluble). A sentence clarifying this has been added in the revised manuscript. Also, the elution profiles from IMAC runs (Fig2) are included as data supporting the “Fusion property” column in Table 2. b. The cassette approach is claimed to be cost effective relative to purchase of short peptides, but with a cost of $60 for a peptide (dependent on locale) the cost of buying the oligonucleotides, cloning, harvesting plasmids, sequencing and then examining the protein produced would be similarly expensive and not that much shorter in time frame. The advantage of the method is the fact that the fused protein is large, which would be prohibitive to synthesis. This should be emphasized. Also, the method could viably be used for internal sequence replacement in a gene coding a protein of interest. Response: The author is indebted to the reviewer for pointing out that longer peptides as well as internal sequence replacements can be made in a cost effective manner. The fact the inserts can accommodate 20 amino acid peptides is now emphasized in the Introduction, as well as in the Conclusion. Additionally, a final sentence is included in the Results and Discussion adding the application of the insertion method to creating internal sequence changes (acknowledging the reviewer). c. To ensure obtained sequence changes are as expected, do the oligonucleotides used require purification eg by HPLC or just desalting? Response: Desalt purity oligonucleotides are used as received from Invitrogen. This is now stated explicitly in the Materials and Methods. d. The author refers to denaturation of the restriction endonuclease. How is this achieved? If by heating, which is the standard practice, how does this affect the insert DNAs? If by addition of chemicals, how is it ensured that ligation is not inhibited? Response: The restriction enzymes are denatured by heating, which is now stated explicitly in the Materials and Methods. Since the insertions are generally successful, it is assumed that heating the DNA does not irreversibly damage it. No additional chemicals which might interfere with ligation are introduced. e. If there is a small change to replace for example a single nucleotide for an amino acid substitution, is it necessary to have the reverse complements strand with this change? I would presume this would give a 50:50 rate of wild type vs mutated sequence in retrieved plasmids. Was this tested? Response: Every mutation introduced in the 5’-to-3’ direction was accompanied by its reverse compliment. This is illustrated for Entries 5 and 6 in Table 1 and in the discussion of these entries. Attempting a mutation by changing only the 5’-to-3’ DNA was never tried. f. Has the author used degenerate oligonucleotides to produce mutated peptides? Please comment here or in discussion. Response: From the manuscript near the end of Results and Discussion: “In this work, generally codons were selected to be most easily expressed in E. coli, with no attention paid to GC content.” This sentence offers the main criterion used for DNA design, namely, that it is most easily expressed in E. coli. Once a DNA sequence was found to work, it was used again. As an example, the DNA coding for the leading poly Gly of Entry 5 is GGA GGT GGA GGT GGA GGT where the alternating codons were picked in order to remove excessive repeats in the DNA (and with no change in Tm). No experiments were done to see if alternate DNA coding for (Gly)6 would perform better, and no mutations were attempted with two sets of degenerate DNA. g. with respect to the conclusion that “close to eight polar residues are required for good solubility when designing 20 amino acid tails.” Since GFP was the only protein tested as a fusion, the statement should be more conservative and state that “close to eight polar residues are required for good solubility when designing 20 amino acid tails appended to GFP”. Response: The change has been made, and also includes the more precise “carboxylate” over the less accurate “polar.” h. Were any analyses performed to determine what proportion of ALL colonies, brightly fluorescent or otherwise, contained plasmids with synthetic DNA inserts? If so, what is the nucleotide sequence error rate for ALL plasmids with inserts? Response: No. i. “Thus, the five segment insertion technique described here has an error rate estimated to be close to 1%.” Because the error rate for ALL plasmids produce was not reported, this should read “Thus, for recombinants with bright GFP fluorescence the five segment insertion technique described here has an error rate estimated to be close to 1%.” Response: The change has been made, but using the following, which is stylistically closer to the manuscript: “Thus, for plasmids sequenced using the colony selection procedure outlined, the five segment insertion technique described here has an error rate estimated to be close to 1%.” j. Overall, the results as described (but not presented) show promise for the versatility of the system and proof of principle. However, the versatility for carrier proteins which do not have a clearly discernible phenotype like GFP is unknown. With no indication of the true successful insert retention and so the true percent insert error rate, the usefulness for rapid selection of recombinant fusion genes is also not known. This should be made clear in this short report. Response: The following passage is included after the 1% error rate sentence: “It should be emphasized the true insertion error rate would be found by sequencing all plasmids from all colonies, and could include a study of which DNA sequences are more prone to errors. Such a study is beyond the scope of this work. The 1% figure used here is meant to suggest a low but still significant error rate, as a caution to researchers contemplating the use of this technique. Additionally, it is unknown how the method will perform with proteins other than GFP, with its convenient green fluorescent phenotype.” Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0307713.r002
10 Jul 2024 Decision Letter - Dhana Govind Gorasia, Editor A convenient five-segment cassette procedure for DNA insertions coding for novel peptides PONE-D-24-14914R1 Dear Dr. Filley, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Dhana Govind Gorasia Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0307713.r003
Formally Accepted
17 Jul 2024 Acceptance Letter - Dhana Govind Gorasia, Editor PONE-D-24-14914R1 PLOS ONE Dear Dr. Filley, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Dhana Govind Gorasia Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307713.r004

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