A protocol for chemical competence in phytopathogenic Ralstonia
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Here, we present a protocol for the transformation of Ralstonia solanacearum species complex (RSSC) strains by calcium chloride-induced chemical competence. We include a step-by-step protocol with media and solution recipes, quantitative results of the protocol's efficacy for four of five tested RSSC strains, and an explanation of the logic underlying the protocol development. The protocol involves sequentially treating overnight cultures with a cold calcium chloride solution and a cold calcium chloride/magnesium chloride/glycerol solution, transforming through alternating heat shocks and ice incubations, and recovering cells via an outgrowth in rich medium.
Article activity feed
-
Thank you for submitting the manuscript revision. I am pleased to inform you that your manuscript has been accepted for publication. This protocol represents a valuable addition to the field, providing a clear, practical and well‑validated method for transforming Ralstonia solanacearum species complex strains.
-
-
Dear Authors, your manuscript has now been reviewed by two experts in the field, who note that the study is clearly and thoroughly presented, with a robust and well‑described methodology. The reviewers consider the work to represent a valuable contribution to the field and a useful resource for the research community. Please address the reviewer comments as outlined to support progression toward publication. I look forward to receiving your revised manuscript.
-
Comments to Author
It is written perfect. The research is important and helpful to the field.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
This manuscript presents a modified protocol for the transformation of members of the Ralstonia solanacearum species complex using chemically induced competence. The authors provide thorough detail of both the experimental protocols used, the steps that were taken to refine and optimize the protocol, and the rationale behind the design of the procedure. The methodology presented is rigorous and clearly presented. The manuscript is well designed to provide a new and useful tool to the Ralstonia research community. Some minor points could be improved to provide more clarity: Line 106: Here, and in the preliminary protocol, you state that the cell treatment step could not be scaled up. Could you provide more detail about how it failed? Were there simply no successful transformants? Line 263: Include …
Comments to Author
This manuscript presents a modified protocol for the transformation of members of the Ralstonia solanacearum species complex using chemically induced competence. The authors provide thorough detail of both the experimental protocols used, the steps that were taken to refine and optimize the protocol, and the rationale behind the design of the procedure. The methodology presented is rigorous and clearly presented. The manuscript is well designed to provide a new and useful tool to the Ralstonia research community. Some minor points could be improved to provide more clarity: Line 106: Here, and in the preliminary protocol, you state that the cell treatment step could not be scaled up. Could you provide more detail about how it failed? Were there simply no successful transformants? Line 263: Include the data for pUFR80 transformations either in Figure 1 or in a supplemental figure Line 307: Include the transformation data for the experiments using more heat shock cycles and varying heat shock temperatures. Seeing how much additional heat shock cycles improved the efficiency would be useful for researchers deciding how to optimize their protocol
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-