High-throughput FastCloning technology: A low-cost method for parallel cloning

Hua Jiang; Fan Meng; Deren Lu; Yanjuan Chen; Guilin Luo; Yuejun Chen; Jun Chen; Cheng Chen; Xi Zhang; Dan Su

doi:10.1371/journal.pone.0273873

Peer Review History

Original SubmissionMay 4, 2022
12 Jun 2022 Decision Letter - Paulo Lee Ho, Editor PONE-D-22-13103High-throughput FastCloning technology: A low-cost method for parallel cloningPLOS ONE Dear Dr. Su, 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 27 2022 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. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: [This work was supported by grants from the National Key Research and Development Program of China (2017YFA0505903), the National Natural Science Foundation of China (31370735, 31670737 to D. S., 81671494 to C. C.), the Technology Department of Tianjin Foundation (19YFZCSN00470), 1·3·5 projects for disciplines of excellence, West China Hospital, Sichuan University (ZYJC18033), the Special Research Fund on COVID-19 of Sichuan Province (2020YFS0010), and the Key Project on COVID-19 of West China Hospital, Sichuan University (HX-2019-nCoV-044).] We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [This work was supported by grants from the National Key Research and Development Program of China (2017YFA0505903), the National Natural Science Foundation of China (31370735, 31670737 to D. S., 81671494 to C. C.), the Technology Department of Tianjin Foundation (19YFZCSN00470), 1·3·5 projects for disciplines of excellence, West China Hospital, Sichuan University (ZYJC18033), the Special Research Fund on COVID-19 of Sichuan Province (2020YFS0010), and the Key Project on COVID-19 of West China Hospital, Sichuan University (HX-2019-nCoV-044).] Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 6. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. 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: The authors describe a fast-cloning method that had been published previously (FastCloning: a highly simplified, purification- free, sequence- and ligation-independent PCR cloning method, 2011), therefore, the technique is interesting but not new, once it was already published. However, in this work, this method was further explored to construct different vectors of expression, and three genes from Mycobacterium tuberculosis were used to evaluate the cloning efficiency of molecular cloning techniques, therefore, extending the previous studies on this technique which may be interesting to be explored for the scientific community. Some questions/comments 1- In Materials and methods, lanes 211-220 say that the amplicon was mixed with an amplified vector. However, since in this case DpnI was not added, and no overhang was created, should some denaturing event carried out to help to promote the base pairing? 2- Fig. 2- I think this figure can be simplified by just adding two vectors since apart from the resistance gene, they are all the same. Maybe two vectors and inside one (pFastB1-6) and the other (pFastB7-12). 3- Fig. 5. Is this gel performed with reduction conditions? Because is not clear why there are 2 bands in the elution, could you explain this a little better? Reviewer #2: The article is well written. Despite not being scientifically original, it brings an elegant and alternative method of cloning in expression vectors. The constructs were analyzed by restriction pattern, but I did not find data that they were sequenced, if any mutation occurs, especially in polycistronics, it can harm the entire genetic construct. It is also not clear what analysis was done on the primers, what are the chances of self annealing, for example? In Figure 3, it would be interesting to place the western blot and/or the negative controls to compare the expression of the proteins. The same goes for figure 5. In figure 2, describe P, apr and Kar. The way the arrow indicates the target for P suggests that the gene would be inserted in this region. The difference between pFASTV1-V12 and pFASTB1-B12 is unclear. In table 1 you could add the size in Kpb of the vectors used. I suggest changing the name of the vectors, as pFAST already exists, they are vectors marketed by Thermofisher for the baculovirus system. ****** 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.0273873.r001
Revision 1
30 Jul 2022 Author Response Dear Editor Paulo Lee Ho, Dear reviewers Thank you for your letter. We were pleased to know that our work was rated as potentially acceptable for publication in Journal POLS ONE, subject to adequate revision. We thank the reviewers for the time and effort that they have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in MS Word. Appended to this letter is our point-by-point response to the comments raised by the reviewers. The comments are reproduced and our responses are given directly afterward in a different color (red). We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. Reviewer #1: The authors describe a fast-cloning method that had been published previously (FastCloning: a highly simplified, purification- free, sequence- and ligation-independent PCR cloning method, 2011), therefore, the technique is interesting but not new, once it was already published. However, in this work, this method was further explored to construct different vectors of expression, and three genes from Mycobacterium tuberculosis were used to evaluate the cloning efficiency of molecular cloning techniques, therefore, extending the previous studies on this technique which may be interesting to be explored for the scientific community. Some questions/comments 1- In Materials and methods, lanes 211-220 say that the amplicon was mixed with an amplified vector. However, since in this case DpnI was not added, and no overhang was created, should some denaturing event carried out to help to promote the base pairing? Answer: Thanks for your question. We use DpnI is to digest the methylated parent DNA templates (if in a plasmid) for PCR amplification, avoid causing false positives after transformation. The lethal gene (ccdB ) was added into our modified vector at the insert region. A successful insertion will completely replace ccdB with the investigator’s insert of interest. Hence correct clones are identified much more efficiently, as those that do not contain the desired insert should not grow. Therefore, it is not necessary to add the dpn1 digestion template. As for how overhang formed, although the detailed mechanism is not known, it is likely that the 3’ exonuclease activity of the high-fidelity DNA polymerase directly creates sticky ends for the overhang of the vector and insert, allowing them to form a circular construct with nicks. 2- Fig. 2- I think this figure can be simplified by just adding two vectors since apart from the resistance gene, they are all the same. Maybe two vectors and inside one (pFastB1-6) and the other (pFastB7-12). Answer: Thanks for your suggestion, we have made correction according to your comments in figure 2. 3- Fig. 5. Is this gel performed with reduction conditions? Because is not clear why there are 2 bands in the elution, could you explain this a little better? Answer: Thanks for your question. Here we make further explanation, in the polycistronic expression system, as the schematic diagram illustrating in figure 4, we generated the co-expression plasmid, Trim6-Trim61- pFastB1, the Trim6 protein fused to a 6×His tag , whereas the untagged protein Trim61 has interaction with Trim6,they can form stable complex, so there are two bands in elution (Fig.5A). By the same token, Epl1,Yng2,Eaf6 can form complex with Esa1, so there are 4 bands in elution(Fig.5C). Reviewer #2: The article is well written. Despite not being scientifically original, it brings an elegant and alternative method of cloning in expression vectors. The constructs were analyzed by restriction pattern, but I did not find data that they were sequenced, if any mutation occurs, especially in polycistronics, it can harm the entire genetic construct. It is also not clear what analysis was done on the primers, what are the chances of self annealing, for example? Answer：Thanks for your question. We used the DNA polymerase contained in MCLAB is Goldenstar T6 DNA polymerase modified by genetic engineering. It has a very high fidelity (60 times of Taq), not easy to produce mutations during PCR. And we sequenced the whole vectors (pSDB1-B12) to ensure the correctness of the vectors’ base sequence. The sequenced data show in supplementary information folder named sequencing data. But we can’t guarantee that mutations won’t be introduced into the construct for other reasons. Considering the cost of sequencing, we only sequenced the insert gene of the three genes from Mycobacterium tuberculosis were used to evaluate the cloning efficiency of molecular cloning techniques, and the constructed plasmid was transferred to BL21(DE3) for protein expression. The results showed that the molecular weight of the expressed protein conformed to the theoretical molecular weight. It is proved that the gene expression element on the constructed plasmid is complete. As for the analysis on the primers, we calculated the Tm value of primer with software Primer 5, optimized the Tm values of high F and high R for vector amplification, and tested that the optimal annealing temperature is 60 ℃ by PCR experiments, won’t cause primer self annealing. The Tm value of primers used for gene amplification will change with different insert gene sequence. The annealing temperature should be adjusted according to specific conditions. Generally, it is appropriate to subtract 5 ℃ from Tm value. In Figure 3, it would be interesting to place the western blot and/or the negative controls to compare the expression of the proteins. The same goes for figure 5. Answer: We appreciate the reviewer for this kind recommendation. In figure 3A,3D and 3F, the negative controls are shown in the first lane, the expression of the proteins are denoted by black triangles according to the expected size. In the figure, the protein expression bands of some vectors are obvious, such as V1, V3, V4, V5, V6, V7, V11and V12 of Mqo. We admit that we cannot guarantee all 12 expression plasmids in the high-throughput plasmid construction system can express the target protein. Our purpose is to screen the best expression vector of the target protein by High-throughput Fast-Cloning technology. We also appreciate your proposal to compare protein expression with the negative controls using Western blot. However, due to the impact of the COVID-19 in Chengdu, we are in control area, resulting in the inability to purchase the corresponding antibodies for WB experiment. We sincerely hope you can understand. As for figure 5, the expressed protein Trim6 (Fig.5A),Eas1(Fig.5C),Lsm7(Fig.5D) fused to a 6×His tag, this tag will binding on Ni-NTA resin, and the target protein can be eluted with high concentration of imidazole, the bands in elution indicates that the target protein is expressed. In figure 5B, GST tag was fused to Trim6, the target protein complex was purified by glutathione affinity chromatography, eluted by reduced glutathione. In figure 2, describe P, apr and Kar. The way the arrow indicates the target for P suggests that the gene would be inserted in this region. Answer: Thanks for your suggestion. P: promoter. Apr: Ampicillin resistance gene. Kar: Kanamycin resistance gene. We revised the figure 2 to make it clearer that the gene will insert between high F and high R. The difference between pFASTV1-V12 and pFASTB1-B12 is unclear. Answer: We thank the reviewer for raising this question. We created a 36-bp DNA adaptor, replacing the original multiple cloning site, which contains many restriction sites in each vector(pFASTV1-V12), to linearize the 12 vectors, we designed a pair of primers “HighF and HighR” according to the sequence of the DNA adaptor. This strategy gives the user the ability to linearize all modified expression vectors by PCR amplification with one pair of primers. For pFASTB1-B12, a lethal ccdB gene expression core box (containing the Cat promoter, the open reading frame of a chloromycetin-resistance gene, and the open reading frame of the ccdB gene) was inserted into pFASTV1-V12 between HighF and HighR, which can eliminate false-positive colonies harboring the template circular plasmid without using DpnI digested the template plasmid. In table 1 you could add the size in Kpb of the vectors used. Thanks for your suggestion, we have added the size of the vectors in table 1. I suggest changing the name of the vectors, as pFAST already exists, they are vectors marketed by Thermofisher for the baculovirus system. Answer: Thank you for your kind reminder. We have changed the name of the vectors pFAST to pSD in the revised manuscript. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0273873.r002
17 Aug 2022 Decision Letter - Paulo Lee Ho, Editor High-throughput FastCloning technology: A low-cost method for parallel cloning PONE-D-22-13103R1 Dear Dr. Su, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. 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: All questions raised in the previous review was properly addressed and I feel that this manuscript is now acceptable for publication. ****** 7. 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 ******** https://doi.org/10.1371/journal.pone.0273873.r003
Formally Accepted
30 Aug 2022 Acceptance Letter - Paulo Lee Ho, Editor PONE-D-22-13103R1 High-throughput FastCloning technology: A low-cost method for parallel cloning Dear Dr. Su: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Paulo Lee Ho Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273873.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .