We report a novel metagenomic library construction and screening method that uses fosmids containing an endogenous gene expression tag. This allows the identification of clones expressing a particular function, such as carbenicillin resistance (CbR), without using a metagenomic strain. Clones encoding CbR can then be sorted using the Substrate Induced Gene Expression (SIGEX) technology24, whereby the SIGEX system identifies induced versus uninduced bacteria. The results show that the SIGEX system significantly increases recovery of clones coding for CbR, while also increasing the number of CbR-conferring fosmids identified by direct selection from the bulk metagenomic library.
The original metagenomic fosmid pCC1FOS-CeuI was used to construct a new vector pMPO579. This was reconstructed to contain the promoterless gfp gene downstream of the vector and metagenomic DNA cloning sites, in order to allow transcription initiated at the metagenomic DNA cloning site to continue through the thnL transcription terminator (Fig. 1). This modification is required for the exploitation of the SIGEX system, whereby the gfp signal generated by the thnL terminator is used to identify transcriptionally active metagenomic clones by fluorescence assisted cell sorting.
To determine the potential of the MPO555 vector to transfer CbR-conferring clones, it was transformed into EPI300-T1 strains carrying various types of plasmids. Six of the resulting specialized strains conferred CbR to 100 mg L-1 carbenicillin. All of these strains contained the blaC gene from E. coli chromosome EPI300-T1 and encoded a class C b-lactamase with or without a short orf immediately downstream of the blaC 3' end, which is most closely related to homologues found in Pseudomonas species.
Triparental matings were performed in which EPI300-T1 carrying the different plasmids was used as the donor, rifampicin resistant (RifR) or nalidixic acid resistant (NalR) derivatives of the same strain were used as the recipient, and DH5a bearing pRK2013 was used as the helper strain. The plasmid pMPO579 was transferred with high efficiency to all of the recipient strains, although its level of expression was kept low by insertion of the constitutive thnF attenuator between the lacUV5p and gene-1 promoters.
To test the ability of pMPO579 to support conjugation, it was further modified by adding the oriT from plasmid RP4 to the unique HpaI site in pCC1FOS-CeuI, to generate the new pMPO561. The thnL transcription terminator was then inserted at the Eco72I site of pMPO579 to yield pMPO580, which is expected to allow transfer of CbR-conferring clones via conjugation with a wide range of E. coli bacterial hosts. The high conjugation frequencies obtained with pMPO580 indicate that the MPO579 vector is well capable of transferring metagenomic clones.
No comments:
Post a Comment