Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli
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Synthetic biology enables the creative combination of engineering and molecular biology for exploration of fundamental aspects of biological phenomena. However, there are limited resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. We developed unigems, a set of ten plasmids that enable classroom-based investigation of gene-expression control and biological logic gates to facilitate teaching synthetic biology and genetic engineering. It is built on a high-copy plasmid backbone and is easily extensible thanks to a common primer set that facilitates Gibson assembly of PCR-generated or synthesized DNA parts into the target vector. It includes two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The set can readily be employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. All comments were satisfactorily addressed.
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Comments to Author
This is a very interesting manuscript focused on use of plasmids and parts for teaching microbiology. Overall, the paper is well-written, with sound methodology and the potential for strong impact in the teaching community. Some minor corrections: Introduction: briefly describe how hands-on learning can improve learning efficiency and student enthusiasm. Conclusions: did you have any feedback from the students? (optional) Has it helped boosting enthusiasm and performance? Section 8.3: what was the volume of the liquid cultures? Line 276: rpm not RPM, be consistent
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 …
Comments to Author
This is a very interesting manuscript focused on use of plasmids and parts for teaching microbiology. Overall, the paper is well-written, with sound methodology and the potential for strong impact in the teaching community. Some minor corrections: Introduction: briefly describe how hands-on learning can improve learning efficiency and student enthusiasm. Conclusions: did you have any feedback from the students? (optional) Has it helped boosting enthusiasm and performance? Section 8.3: what was the volume of the liquid cultures? Line 276: rpm not RPM, be consistent
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
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community. The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
This paper describes the development of a set of plasmids that can be used to teach synthetic biology and gene regulation using fluorescent and olfactory reporters. The strengths of the system are the easy ability to manipulate and alter the plasmids allowing for the creation of new constructs and the variety of established promoters and reporters being used. Overall the paper is well written and the methods and results well described and I have no doubt that the Unigems plasmid system will be an incredibly useful resource for educators and those involved in outreach alike. I have made a few comments below which I think would be useful to provide a little more context for those interested in using the system and a little more clarity to some sections but overall I think the paper gives a nice detailed …
Comments to Author
This paper describes the development of a set of plasmids that can be used to teach synthetic biology and gene regulation using fluorescent and olfactory reporters. The strengths of the system are the easy ability to manipulate and alter the plasmids allowing for the creation of new constructs and the variety of established promoters and reporters being used. Overall the paper is well written and the methods and results well described and I have no doubt that the Unigems plasmid system will be an incredibly useful resource for educators and those involved in outreach alike. I have made a few comments below which I think would be useful to provide a little more context for those interested in using the system and a little more clarity to some sections but overall I think the paper gives a nice detailed overview of a very useful set of molecular tools for teaching and outreach purposes. 1) Line 51 - I couldn't get the "addgene" link to work. Could the authors check this is active (and apologies if this is a limitation on my part). 2) Line 70 - Should this be "which provide" rather than "which provides"? 3) Line 84 - Should the plasmid p007KanGFP not be p007AmpGFP? 4) Line 137 - Should this be "specific to this gene" rather than "specific to these genes"? 5) Line 160 - Figure 3 - The lack of a significant difference between the strong and weak promoter using RFP is mentioned in the discussion to be due to the readings being taken at a non-optimal wavelength. I think the authors need to clarify this in the results section and mention this limitation for the data shown at the time the data is shown. 6) Line 171 - Figure 4 - This figure shows inducibility of the system using arabinose and IPTG. The arabinose was tested from 0-5% and the IPTG from 0-5mM. The authors comment "pBAD exhibiting much more prominent inducibility than T5-pLacO" is a little vague. It might be useful to point out that the T5-pLacO produces a much more dose dependent and gradual response than the pBAD in this system. I would have expected the pBAD to be better for dose dependent induction but either way this is a useful difference between the two systems and would be an important consideration for those choosing which inducible system to use for a specific teaching or outreach application. I think a few more lines on the difference in inducibility between the two systems would be helpful. 7) Line 181 - the phrase "is clearly visible and in the presence of both inducers" is a little confusing. I would suggest "is clearly inducible in the presence of both inducers" or another alternative version. 8) Line 188 - The section on the olfactory construct - The inclusion of an olfactory construct is a nice addition to this toolkit. Its clear this is expressed at stationary phase but it would be useful to clarify whether the odour is detectable in plate as well as broth culture. 9) Line 196-198 - This sentence in whole reads a little strange. I would suggest replacing the final phrase "rather than deliver precise quantitative output" with "rather than being designed to deliver a precise quantitative output" 10) Discussion - The authors are describing an incredibly useful toolkit and have mentioned that it could be used for outreach as well as educational purposes. It would be very useful to include a paragraph clearly outlining the equipment requirements for detection of fluorescence from this system and the overall tractability of the system which I suspect is one of its strengths. Might be useful to factor in here the whether the RFP or GFP is more tractable and the limitations depending on whether you an excite at a specific wavelength. They mention some of these reporters are visible by the naked eye and it would also be useful to mention here whether for outreach purposes how visible they would be on plates. Effectively a little more information on the tractability of the system for the end user would be very useful 11) Line 241 - Can the authors also include the conc of ampicillin used as well as they do describe a plasmid with an amp cassette.
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
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