Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Abstract

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinoseinducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

Mining the Metabiome: Identifying Novel Natural Products from Microbial Communities

“…(Gregory et al, 2003; Kuhstoss and Rao, 1991). For transfer of libraries into diverse alpha-, beta-, and gammaproteobacterial species, several RK2-derived vectors have been recently developed (Aakvik et al, 2009; Kakirde et al, 2011). The utility of these vectors was demonstrated by parallel functional metagenomic library screens carried out in Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans, using a single broad-host-range vector.…”

Metagenomic small molecule discovery methods

“…(51, 52) More recently, RK2-derived vectors have been constructed to allow movement of libraries into diverse alpha, beta, and gamma proteobacterial species. (53-55) Creating libraries in shuttle vectors allows for conjugative transfer of eDNA into a wide range of hosts, including bacteria belonging to biosynthetically-rich phyla like Streptomyces and beta-proteobacteria. (56)…”

Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

“…There are cloning vectors based on several broad-host-range replicons, e.g. RK2 of IncP-1 (Aakvik et al, 2009;Blatny et al, 1997;Crouzet et al, 1992;Ditta et al, 1985;Dombrecht et al, 2001;Kakirde et al, 2011;Santos et al, 2001;Scott et al, 2003;Thorsted et al, 1998), RSF1010 of IncQ (Bagdasarian et al, 1981;Chistoserdov and Tsygankov, 1986;Coppi et al, 2001;Frey, 1992;Gambill and Summers, 1985;O'Sullivan et al, 2010;Parales and Harwood, 1993;Priefer et al, 1985;Rangwala et al, 1991;Sharpe, 1984), pBBR1 of IncA/C (Kovach et al, 1994;Kovach et al, 1995) or rolling circle replicon originated from pWV01 (Bryksin and Matsumura, 2010).…”