Rapid approach for cloning bacterial single-genes directly from soils

Abstract

Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of cloning strategy in this study. Five GS genes were directly cloned from soils using degenerate primers with two steps of nested polymerase chains reactions. The genes showed 94 to 99% amino acid identities to the homologs in the known database, and encoded proteins affiliated to GS I and GS II families, respectively. All the five genes could rescue the growth of Escherichia coli glutamine auxotroph mutant ET6017 in minimum medium (ammonium chloride was sole nitrogen source in this medium). This study develops one rapid approach for cloning bacterial single-genes directly from soils. Comparing with the conventional strategies for gene cloning from complex environmental samples, this method did not need making genomic library and isolating target genes from large amount of library clones. This approach distinctively demonstrates its advantages of rapidity and effectiveness particularly when it aims at cloning short single-genes that had known homologs in all kinds of nucleic acid databases.

Keywords: Gene cloning, soil, glutamine synthetase, nested PCR, single-gene

African Journal of Biotechnology Vol. 12(32), pp. 5029-5034