Tuesday, October 31, 2006

Q and A on BACs and Metagenomics. Why large inserts?

Question from student (edited):

I am reviewing the Soil Metagenome paper that we reviewed in class (Rondon et al., 2000, Appl. Environ. Microbiol. 66: 2541-2547), and have a written note claiming that there are two important reasons to clone large DNA fragments into the BAC vectors. I can only think of one reason, which is that many soil microbes produce important bioactive compounds, and genes required for production along with regulatory genes are often clustered in one continuous segment on the chromosome called an operon. A large fragment of DNA inserted into BAC plasmid would increase the likelihood that the entire operon included. Is this correct, and is there another reason why having large DNA fragments in the BAC plasmids is important?

Answer:

Yes, you are correct, and yes, there is another reason. Large inserts mean that each clone (Escherichia coli clone line) is likely to contain genes encoding for multiple activities that may be of interest. This means that fewer clones need to be maintained than if the inserts were smaller, i.e., the same amount of metagenomic information is maintained in fewer clones (or more metagenomic information can be maintain in any given number of clone lines). Using screening assays for different activities, the same amount of metagenomic material can therefore be interrogated for the presence of specific activities while manipulating fewer cultures.

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