Sequence and origin of the Streptomyces intergenetic-conjugation helper plasmid pUZ8002
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
Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence.
Article activity feed
-
-
Thank you for addressing all reviewer comments, I am confident that this will be a useful resource for the community.
-
-
This work is well presented and is a useful resource for the research community. The reviewers have only minor comments that should be addressed. Please also ensure that sequencing reads are publicly available.
-
Comments to Author
In this manuscript, Larcombe et al. describe the construction and sequence of pUZ8002, a widely used helper plasmid for mobilisation of genetic material from E. coli into Streptomyces species. The authors note that there is already a pUZ8002 sequence available in Genbank, however they note a handful of differences between their sequence and that already available, which is to be anticipated because the unstable genetic nature of E. coli ET12567, which is genetically unstable. The work is well presented and of interest to the community - it clarifies the scientific record for pUZ8002 and provides another genetic sequence for the widely used construct. The methods are robust, and the conclusions are accurate. I believe these data will be of interest to the community it serves and only have minor …
Comments to Author
In this manuscript, Larcombe et al. describe the construction and sequence of pUZ8002, a widely used helper plasmid for mobilisation of genetic material from E. coli into Streptomyces species. The authors note that there is already a pUZ8002 sequence available in Genbank, however they note a handful of differences between their sequence and that already available, which is to be anticipated because the unstable genetic nature of E. coli ET12567, which is genetically unstable. The work is well presented and of interest to the community - it clarifies the scientific record for pUZ8002 and provides another genetic sequence for the widely used construct. The methods are robust, and the conclusions are accurate. I believe these data will be of interest to the community it serves and only have minor corrections for the authors to consider. Minor corrections: Lines 38-40: Consider using a more current reference for this. The one that is most often used to support this statement is PMID: 32162523. Line 45: I agree with the statement, however, consider providing published examples illustrating it is in fact true. Line 61: Change Rk2 to RK2 Line 100: Change table 1 to Table 1 Line 101: Use the prime symbol instead of the apostrophe. Line 101-103: Consider rewording the start of this sentence to, "Destruction of the nic site prevents…" Line 107: Given the discussion focuses on plasmids instead of organisms, consider replacing in with for, i.e. "For pRK221761…" Line 109: Should oriT be in lowercase italics here instead? Line 139: I an unclear on what the journal guidelines are, however for many publishers it is customary to only spell out numbers when they are less than 10 or start sentences, so 30 instead of thirty, here. Line 142: Depending on the journal format, change this to the beta symbol rather than spelling it out. Line 143: Amend the text as appropriate for, "hypothetical protein with of unknown function". Line 145: Comma required in 2939 bp and prime symbol instead of apostrophe. Line 152: See above, consider amending to "Additionally, for pUZ8002…" Line 161: Use the mu symbol instead of a u. Line 262: The significance of the 250RPM is unclear, consider removing. Table 1: Specifying (bp) in the column header of size, and also consider describing this as length instead of size. Lines 268-173: Consider reworking the summary section to be a little more concise. In addition, I would argue that the main output of the study is that you have indeed preserved the historical record in Streptomyces genetics, so you should state so proudly, rather than hoping that is what you have done.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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 full annotated sequence of plasmid pUZ8002, a conjugative plasmid routinely used to transfer plasmids into actinomycete strains. Although a previous pUZ8002 sequence is publicly available in GeneBank, there is no prior publication that reports the complete sequence and details its construction process. This manuscript provides access to pUZ8002's complete sequence, construction methodology and plasmid features which is very relevant to the actinomycete research community, provides very useful information and should help troubleshooting of intergenetic conjugations between E. coli and actinomycete strains in future. Overall, the manuscript provides a clear and well written description of the construction and highlighted pUZ8002 features which is easy to follow. All relevant …
Comments to Author
This manuscript presents a full annotated sequence of plasmid pUZ8002, a conjugative plasmid routinely used to transfer plasmids into actinomycete strains. Although a previous pUZ8002 sequence is publicly available in GeneBank, there is no prior publication that reports the complete sequence and details its construction process. This manuscript provides access to pUZ8002's complete sequence, construction methodology and plasmid features which is very relevant to the actinomycete research community, provides very useful information and should help troubleshooting of intergenetic conjugations between E. coli and actinomycete strains in future. Overall, the manuscript provides a clear and well written description of the construction and highlighted pUZ8002 features which is easy to follow. All relevant data is available to the reader either via GeneBank accession numbers or Figshare. The last sentence of the introduction (lines 75 to 77) says: "This work aims to verify the sequence of pUZ8002 from our commonly used laboratory strains of E. coli, detail the process for the construction of pUZ8002 and determine the differences from RP4." RP4 is mentioned four more times after this in the manuscript. However RP4 is not mentioned prior to this sentence. I suggest that the authors clarify why they want to compare pUZ8002 to RP4 by briefly adding a few lines introducing RP4 and its relation to pUZ8002.
Please rate the manuscript for methodological rigour
Very good
Please rate the quality of the presentation and structure of the manuscript
Very 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
-