Abstract:

The construction of large-fragment metagenomic library helps to discover large-fragment functional genes and analyze their organization and function. In this paper,first,three methods,namely the CTAB method,the kit and the agarose-embedding method,were used to extract metagenomic DNA from activated sludge,and DNA fragments larger than 23 kbp were extracted by the agarose-embedding method. Then,the metagenomic DNA was ligated into pCC1FOS vector to construct a Fosmid library consisting of 5280 clones with an average insert fragment size of 35 ～40 kbp,which covered about 200Mbp of metagenomic DNA. Finally,one clone exhibiting amylase activity was rapidly screened from 200 randomly-picked clones via the activity-based screening. It is thus concluded that the Fosmid library constructed from activated sludge is feasible in the screening of novel functional genes by activitybased methods.

Key words: activated sludge, metagenomic DNA, Fosmid library, activity-based screening, amylase