I have been pondering the idea of using the construction of a cosmid genomic (or metagenomic) library for the identification of novel genes in gram positive bacteria. With the research I have done, I get the impression that this technique is more common to gram negative bacteria.
Having said that, does anyone know of any studies which have successfully modified this technique for gram positive strains?
Hi Adam,
I agree with your doubt that majority of the cosmid library studies has been conducted with gram negatives. Yet, as far as the cosmid library construction method is concerned, I have not found that this method is specific for the gram negative bugs. Thus, you can try to prepare the cosmid library from the gram positive without any special modifications. You can find good references in the following literature for the clone library preparation.
Adam, I am presuming you mean that you want to construct a cosmid library to screen in gram positive bacteria. One could prepare such a library in E. coli and then transfer to the bacterium of your choice, but you would need to create a cosmid shuttle vector first before building the library and then would be limited by your ability to introduce the library into your organism.
The advantage to cosmids are that since they can carry more DNA per clone it becomes easier to build a more complete library from a large genome organism. That isn't such a problem with a bacterial genome. So it would probably just be easier to prepare a standard genomic library from your bug in whatever plasmid is most appropriate, and not worry about a cosmid. The exception might be if you are looking for very large operons or linked pathways that might not clone readily in a normal library.
Hello Michael, thank you for your response. I have found with normal plasmid-based genomic library is that there is a plasmid size bias. Hence, larger plasmids (with larger inserts) tend not to be taken up by the screening host as effectively; though the idea is something I have considered. The problem I face with cosmid-based genomic libraries is the following: I need a cosmid vector which can replicate in both gram positive and negative (since I would make the library in e. coli and use conjugation to transfer it over to a gram positive host).
Knowing of an established method, for which someone as identified a suitable cosmid plasmid would be beneficial. Otherwise, I would need to experiment with establishing a standard protocol for gram positive cosmid library constructions.
If you have an existing shuttle plasmid that works well for you, then adding a cos site to that construct is fairly straightforward and is the only thing you need to convert it to a cosmid.
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