GEM-Gate: A Low-Cost, Flexible Approach to BioBrick Assembly

Round 1

Reviewer 1 Report

This is an interesting short paper describing a general method to convert BioBricks from the Registry of Standard Biological Parts into PCR products which can be assembled using Golden Gate-type methods, which are much faster than BioBrick assembly, since they allow assembly of multiple parts in a single reaction. The procedure is simple and well described. The basic idea should be obvious to anyone skilled in the art, but having said that, the specific primers used, and the results obtained with real BioBricks, may well be useful to others, especially student teams competing in the International Genetically Engineered Machine competition (iGEM), who may not have the resources for more complex approaches. As such, I think that, with minor modifications, this is a useful contribution to the literature.

My main criticism is that some of the results obtained are described in fairly vague terms, and I would like to see more detail added. For example, lines 74 to 76 say 'Unfortunately, this version of the primers were not as robust and sometimes failed to amplify the target'. One example is shown in Figure 2, but I would prefer (if the editors agree) that a complete list should be provided of all the BioBricks that were tested with each version of the primers, and whether or not amplification was successful in each case, and whether shorter products suggesting binding to internal scar sequences (as described in lines 93-94) was detected. This might be in Supplementary Information if the authors prefer not to include it in the main text. Likewise in lines 117-118 the authors mention 'recalcitrant BioBricks'; which were these? And in line 121 the authors state that a two step strategy was 'successful in amplifying all BioBricks tested'; how many BioBricks were tested, and which ones?

Another issue is that the Results (lines 63 to 129) only describe amplification of Parts; however, the Discussion (lines 131-138) describes several different assembly experiments, which are not mentioned in the Results or Methods at all. (In some cases, such as the reverse orientation assembly, it is not quite clear whether such experiments were actually performed or are only considered to be possible in principle using this method.) If these assembly experiments are to be included in the paper (as they certainly should be), then, in my opinion, the experiments and the results should be properly described in the main text of the paper in a suitable level of detail. Some of this material is present in the Supplementary information and could easily be moved.

Likewise the material presented in Discussion lines 139-154 is arguably more appropriate to the Results.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The communication from Bower et al., intent to demonstrate the use of selected primers to amplify and DNA from iGEM registry. I appreciate the effort keeping in mind that such inexpensive demonstration can ease the understanding of undergraduates to expand their learning of synthetic biology methods. But my main concern is regarding this article is related to real application and level of novelty of this article. There are plethora of article available which exploits not only golden gate assembly but other methods like Gibson assembly, gateway cloning, Loop assembly etc. The commercial kits for these methods are available with positive control that are employed at Universities to develop a sense of learning among undergraduate students to understand the field of molecular biology so in that sense I do not see the higher reach of this method for a wider group of scientists and students.  Nevertheless, the approach is adequate but still there are lot of caveats that must be addressed before publication. Here are few comments for authors to consider:

• As I mentioned, I do not see wider application of this method compared to already available methods. To improve the novelty of this study the authors may consider to show the applicability of this method by cloning the same gene using their Universal Golden Gate method and other methods including conventional Golden Gate (Gibson etc.). If they can demonstrate that their approach is more advanced (by comparing pros and cons) and have wider applicability. It will definitely improve the novelty of this method.
• The title Universal Golden Gate appears misleading, as it can amplify only handful of genetic parts which are part of registry. The reader can understand if it can be applied to any gene cloning (atleast this is what I thought when I read the title).
• Golden gate assembly is preferred since it allows assembly of many gene products in single step. Does the Universal Golden Gate can be applied for that?
• Authors demonstated the amplification and assembly of many genes, they can consider of showing expression of genes after assembly to prove that after amplification the gene expression works and their approach is not contributing any adverse effect on expression profile for any gene.
• It is really hard to understand the article the way it is written and correlate all the information presented in the supplementary to the main text. I would appreciate more pictoriual representation of this method to ease the understanding for the reader specially for those who are not used to Golden Gate assembly.

Considering all the points mentioned above, I am not recommending this article for publication in the current state and suggest a major revision of the presented work with a clarification on the benefit of using Universal Golden Gate over already available conventional Golden Gate method more robust examples of their application.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I appreciate the attempt made by the authors to provide point to point response to most of the comments made by my in my previous review. For short communication, the approach is appropriate and steps are well executed but still my major concern is related to novelty of the study.

I do consider that authors were not able to perform additional experiments (GFP measurement) to prove validation of the constructs but I believe having those results are a must to establish the functional validation for any molecular biology related approach for a proof of concept study. The authors may request the editor to provide more time to perform these experiments and can resubmit the manuscript that I am happy to review again.

Author Response

Reviewer 2 (round 2)

“I appreciate the attempt made by the authors to provide point to point response to most of the comments made by my in my previous review. For short communication, the approach is appropriate and steps are well executed but still my major concern is related to novelty of the study.

I do consider that authors were not able to perform additional experiments (GFP measurement) to prove validation of the constructs but I believe having those results are a must to establish the functional validation for any molecular biology related approach for a proof of concept study. The authors may request the editor to provide more time to perform these experiments and can resubmit the manuscript that I am happy to review again.”

After a great delay, we believe we have sufficiently addressed this concern. We have done so by demonstrating assembly of at least three fragments, representing GFP, RFP, SacB, LacZ, or KanR in different combinations, by showing the subsequent expression of these genes (Fig S10). Moreover, we used a diagnostic PCR to verify that the three fragment assembly was successful (Fig S11).

In order to obtain this result, a number of other modifications were necessary which lead to additional insights now in the manuscript. It was discovered that some primer-template mismatches with the V6 primers were being repaired by the high fidelity (i.e. proof-reading) polymerase we employed. We now present this evidence in the paper (Fig S7). This represents a phenomenon that is not widely reported on – indeed, our conversations with technical support staff from New England Biolabs revealed that they know of the potential for such a result, but only from their own private data, and were unaware of the single study that systematically explored this issue (which we now cite in the manuscript). This necessitated a redesign in the primers, leading to the V7 series, which we show to be more robust in PCR and not prone to repair by our chosen polymerase (Fig S8).

In addition, we experienced some delays due to several unforced errors. These includes using primers with incorrect overhangs or failing to include one of the fragments in the reaction. These assembly reactions, when transformed into bacteria, produced little to no colonies. Whatever colonies we did obtain tended to be some carryover template plasmid from PCR or some unwanted, rare side-product from the assembly. Only once we had corrected for these mistakes did we get robust assembly and transformation, with orders of magnitude more colonies and over 90% of colonies harboring the correct construct. Although we would have preferred a more direct path to a successful result, these errors inadvertently provided a negative control that helps reinforce the assembly strategy we are proposing. These observations are briefly mentioned in the revised manuscript.

Some additional minor edits were made to help clarify and simplify some of the images and text.

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Thank you again for considering this response and the revised manuscript. Please let me know if there is any other information I can provide.

Sincerely,
Devin Camenares, Ph.D.

Author Response File: Author Response.pdf