Complete sequences of pIJ101-based Streptomyces-Escherichia coli shuttle vectors

  1. Katelyn V. Brown
  2. S. Eric Nybo

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Abstract

High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces–Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces–E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.

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  1. Access Microbiology

    Thank you very much for submitting your revised manuscript to Access Microbiology and introducing the suggested corrections. I am pleased to inform you that it has now been accepted for publication. Congratulations!

  2. Version published to 10.1099/acmi.0.000893.v3 on Access Microbiology
  3. Version published to 10.1099/acmi.0.000893.v2 on Access Microbiology
  4. Access Microbiology

    Thank you very much for submitting your manuscript to Access Microbiology. It has now been reviewed by three experts in the field, whose comments are found below. All reviewers consider this a valuable contribution to the field, however they have some concerns that need amendment. Please do the suggested corrections and pay special attention to the comments referring to raw data availability and availability of bioinformatics/assembly pipelines and protocols. Please provide a revised version of the manuscript (along with a tracked-changes version) and a point-by-point version of the reviewer comments within 1 month.

  5. Access Microbiology

    Comments to Author

    In this manuscript, Brown and Nybo report the sequencing of two E. coli-Streptomyces shuttle plasmids that have been widely used for decades. The authors note a handful of discrepancies between the published vector maps and the complete plasmid sequence, which while they do not impact the functionality of the plasmids, they certainly would limit the ability to clone/assembly DNA using more modern techniques like Gibson and Golden Gate assembly. The Genbank accessions for the plasmids are live and I believe they will make for a useful resource for the community. I have minor typographical errors to address only. Line 13 (abstract): Rephrase this, "Industrially relevant actinomycetes are interested in producing …" to avoid suggesting that the actinomycetes are 'interested' in anything other than reproducing. Lines 43, 46, 48: De novo should be in italics. Lines 76 and 77: LB traditionally stands for Lysogeny Broth. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC321500/pdf/1119.pdf Line 94: Some information needs to be given for UltraCycler v1.0; is there a pre-print that you can site or a URL you can link in? Line 103: Italics for i.e. and comma afterwards. Lines 156-159: The author contributions section is a different font type to the rest of the manuscript.

    Please rate the manuscript for methodological rigour

    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

  6. Access Microbiology

    Comments to Author

    Here the authors have detailed the sequence of two vectors used in the field of Streptomyces research. Details of the methodology behind the sequencing could be expanded on but the final assembled sequences are already made publicly available and the results are helpfully annotated. The differences between the sequences and previously available maps are made clear. The paper is clear to follow and key findings are plainly detailed. Literature analysis details the history of sequencing methods and explains why a complete and accurate sequence for commonly used lab vectors is essential for cloning workflows. More information could be provided about the use of these vectors in the Streptomyces field which will give context to why the work is important. I would also add that the work is important and will be a great resources to researchers using these vectors in the future! My suggested changes are detailed below: 1) Line 13: "actinomycetes are interested in" sounds like anthropomorphism of the actinomycetes. Suggest rewording to: "Industry are interested in using actinomycetes to produce keratolytic enzymes and various biologically active natural products". 2) Line 18: Suggest removing the words "and mapping" as you say you use de novo assembly later in the text. 3) Lines 20-22 : Reword to: "containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication." As origin of replications are not generally genes that encode proteins. 4) Lines 22-24: Same as point 3. 5) Lines 27: Text refer to the vectors as "expression vectors" here but "shuttle vectors" in title and in other places. Consider keeping it consistent although they are both. 6) Line 57 Suggest rewording to "cloning via restriction digest and ligation". 7) Line 66: Insert references for these two vectors. You use the papers detailing the creation of the vectors later in the text but multiple references would be good as vectors are described as "popular" and "broadly used". 8) Line 76: Missing word: "was grown in lytic". 9) Lines 94-95: Should Brian Seed and Huajun Wang not be listed as authors for this work if they performed the sequencing personally? Additionally, methods are not complete enough to allow someone to replicate for example what settings were used for this assembly? Are the assembly details publicly available or should the code be published for open access? 10) Line 98: I assume "Sequencing and analysis of pEM4 and pUWL201" is the subheading and could be italicised as with the material and methods subheadings to keep formatting consistent. 11) Line 120: The location of this additional XbaI site could be detailed in the text (ie. XbaI at position 510) as other features are covered by table 1. 12) Table 1: pEM4 is listed as not having pIJ101 and having pIJ702 contrary to line 22 where it is listed as having pIJ101. 13) Table 1: Both vectors are listed as having ColE1 origin yet in lines 112-113 the origin is called pMB1 from pUC19. pUC19 is also listed as the origin in lines 21 and 24. 14) Figure 1: As above ColE1 is listed for both vectors. Additionally, it is labelled "origin" in pEM4 and "ori" in pUWL201. General points: 15) Some information about the uses of these vectors in the introduction would help explain the importance of having complete sequences for researchers. This is alluded to in the abstract where industrial interest in actinomycetes for production of natural products Is mentioned but the role of shuttle vectors in studying and improving these functions should be expanded on in the introduction. References demonstrating researchers using the vectors as mentioned in point 7 should help with this. 16) Check the journal requirements but a list of abbreviations may be required (bp, MCS etc.). 17) Check if raw sequencing reads needs to be made available as well as complete assemblies. -

    Please rate the manuscript for methodological rigour

    Satisfactory

    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

  7. Access Microbiology

    Comments to Author

    This short paper presents some useful vector sequences that may be of interest / utility to the Streptomyces research community. It is generally well written and clear - while I might have liked a little more detail in places, I can appreciate that the authors want to be concise. I don't think any major changes are required before publication, but have made some minor suggestions / comments as listed below. Line 1 (Title). I don't think the use of next generation technology is particularly noteworthy here - I would suggest "Complete sequences of... ". Lines 12-13. "Industrially relevant actinomycetes are interested in producing" - the actinomycetes are not interested in anything - please rewrite. Line 18. "and mapping" seems a little vague and i suggest might be better deleted? Lines 39-46. I'm not sure if all of this is needed - NGS is such a widely used and common technique nowadays that I don't believe the basics need explaining as background. Also I'm not sure it is meaningful to contrast "De novo sequencing" and "Sanger sequencing" as you do here. If "de novo" refers to sequencing a novel (plasmid) genome where there is no reference sequence available, then this can also be done via Sanger methods. Line 55. maybe useful to introduce pIJ702 here (or somewhere) as source for ori of pEM4 as in Table 1? Line 76-77. I think LB more correctly referred to as "lysogeny broth" (see e.g. the confusingly titled advice form ASM here: https://asm.org/getattachment/5d82aa34-b514-4d85-8af3-aeabe6402874/LB-Luria-Agar-protocol-3031.pdf). Might be useful to specify Miller / Lennox / Luria formulation? Lines 93-95. Would it be useful to state coverage here? Also, for "(Brian Seed and Huajun Wang, unpublished)" is it possible to provide any more information or a citation? Line 106. Would it be more accurate to say "additional 1089 bp"? Lines 108-110. Are these exact values? If not then why not be exact? If they are then why not indicate where the extra bp occurred? Line 115. Why not start with a summary description of the plasmid as given for pEM4 in previous paragraph? i.e. is it also a "high-copy number, E. coli-Streptomyces spp. shuttle vector"? Line 118. As you have done for pEM4 above, why not explain where this sequence originates from? Line 119. "Sequencing of the multiple cloning site..." I found this a little unclear - "additional" compared to a previous published map? Is there still the second XbaI site that ermE*p was cloned into, or was that destroyed during cloning making this site unique? Please clarify. Table 1. Italic font for bla. tsr, ermE*p. Figure 1. "Plasmid maps for pEM4 and pUWL201" i,e. remove extra s from "Plasmids". Gene names should ideally be in italics. Is it worth briefly explaining f1 origin + fd terminator in text or figure legend? Legend should explain colour-coding of genes. References. Species names should be in italic font.

    Please rate the manuscript for methodological rigour

    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

  8. Version published to 10.1099/acmi.0.000893.v1 on Access Microbiology