MPO555 is a metagenomic vector that allows transcription of metagenomic DNA through two distinct promoters, one from the T7 RNA polymerase gene and the other from the psal promoter of the lambda phage. Transcription from these two promoters is mediated by a unique N anti-termination system that provides processive termination of transcripts. Using this versatile system, we successfully screened metagenomic DNA in the specialized E. coli strains MPO553 and MPO555, both of which produce the necessary N anti-termination protein.
Metagenomic libraries have recently become available that provide a large amount of environmental genomic information1. However, the functional screening of such vast amounts of metagenomic DNA is hampered by the inability to reliably evaluate the function of individual genes. In addition, the transcription of long stretches of environmental DNA often requires the use of broad host-range expression systems that allow the transcription to proceed through any transcriptional terminators present in the environment 2.
The MPO555 strain was constructed by inserting into its trg locus a DNA fragment containing the lacUV5 promoter, the attenuator nasF and gene-1 from the E. coli T7 phage (Fig. 2). This strain produces constitutive levels of T7 RNA polymerase, whose expression is kept low by the nasF attenuator inserted between the lacI repressor and gene-1. The blaC gene of the T7 phage encodes a class-C b-lactamase that is capable of conferring resistance to carbenicillin and other beta-lactam antibiotics. The 103 amino acid open reading frame of blaC contains a 4 nucleotide overlap with the psal gene 3' end, suggesting translational coupling of the blaC mRNA to psal protein.
Induction of gfp expression in MPO555 with arabinose or salicylate showed that psal in MPO555 confers the required function in these conditions. Increasing the plasmid copy number in MPO555 resulted in a modest increase in gfp expression, but much higher levels of expression were obtained in the presence of both arabinose and salicylate (Fig. 3). These results show that the psal promoter in MPO555 is functional and supports transcription of metagenomic DNA through a wide range of activators.
To test whether transcription of cloned metagenomic DNA through the thnL terminator could proceed through any transcriptional terminators in the environment, the thnL terminator from Sphingomonas macrogoliitabida was inserted into the Eco72I site in pMPO579 to generate pMPO580. Induction of gfp expression in pMPO580 with arabinose and salicylate showed that thnL is capable of functioning as a transcriptional terminator, indicating that this modified vector should support successful screening in the specialized strains MPO553 and MPO555. This result is in agreement with the finding that both of these specialized strains produce the requisite activator NahR and the antitermination protein N, as expected
No comments:
Post a Comment