Design and validation of a transposon that promotes expression of genes in episomal DNA.

Clicks: 287
ID: 85216
2020
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Abstract
Functional metagenomics, or the cloning and expression of DNA isolated directly from environmental samples, represents a source of novel compounds with biotechnological potential. However, attempts to identify such compounds in metagenomic libraries are generally inefficient in part due to lack of expression of heterologous DNA. In this research, the TnC_T7 transposon was developed to supply transcriptional machinery during functional analysis of metagenomic libraries. TnC_T7 contains bidirectional T7 promoters, the gene encoding the T7 RNA polymerase (T7RNAP), and a kanamycin resistance gene. The T7 RNA polymerase gene is regulated by the inducible arabinose promoter (P), thereby facilitating inducible expression of genes adjacent to the randomly integrating transposon. The high processivity of T7RNAP should make this tool particularly useful for obtaining gene expression in long inserts. TnC_T7 functionality was validated by conducting in vitro transposition of pKR-C12 or fosmid pF076_GFPmut3*, carrying metagenomic DNA from soil. We identified transposon insertions that enhanced GFP expression in both vectors, including insertions in which the promoter delivered by the transposon was located as far as 8.7 kb from the GFP gene, indicating the power of the high processivity of the T7 polymerase. The results gathered in this research demonstrate the potential of TnC_T7 to enhance gene expression in functional metagenomic studies.
Reference Key
mongu2020designjournal Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Monguí, Alvaro;Lozano, Gabriel L;Handelsman, Jo;Restrepo, Silvia;Junca, Howard;
Journal journal of biotechnology
Year 2020
DOI
S0168-1656(20)30008-0
URL
Keywords
functional metagenomics gfp expression t7 promoter transposon mutagenesis

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