Thursday, April 29, 2010

Identification of suitable hosts for small-molecule functional metagenomics

The majority of soil-dwelling bacteria cannot be cultured with standard microbial culture methods, and represent an untapped reservoir of novel small molecules that are key components of biosynthetic pathways. Functional metagenomics is one approach to solving this problem; however, screening methods are limited by their dependence on a host organism to facilitate the expression of genes from environmental DNA (eDNA).  To overcome the limitations of relying on a single host organism, Craig et al.* examined six unique Proteobacteria hosts for functional metagenomic screening. To construct  soil eDNA libraries, they blunt-ended the isolated DNA with the End-It™ DNA End-Repair Kit, ligated it into a cosmid vector, packaged the DNA using MaxPlax™ Lambda Packaging Extracts, and transformed TransforMax™ EC100 Electrocompetent E. coli. The eDNA libraries were then used to transform each of the six hosts being studied, and the resulting libraries were screened for antibacterial activity, or altered pigment or morphology. Cosmid DNA from selected colonies was electroporated into TransforMax™ EPI300 Electrocompetent E. coli, and each cosmid clone was analyzed by 454 sequencing.

From this initial broad-host-range functional metagenomic study, Craig et al. demonstrate that some surrogate expression hosts will be better suited to using foreign genetic material than others. They conclude that continued exploration of phylogenetically diverse bacteria as hosts for functional metagenomic screening is likely to identify additional strains that will be suitable for eDNA functional metagenomics.*Craig, J. et al. (2010). Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria Applied and Environmental Microbiology, 76 (5), 1633-1641 DOI: 10.1128/AEM.02169-09


MikeS said...

I'm not familiar with the EC100 strain--what's the max size cosmid that you've successfully transformed?

epibio said...

We've used the EC100 and EPI300 cells with BACs around 150-160 kb, and customers have used even larger BACs successfully. However, this size range is dependent on the BAC and the quality of the DNA prep.