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

• Published in: Journal of biotechnology 2020-02, Vol.310, p.1-5
• Main Authors: Mongui, Alvaro, Lozano, Gabriel L., Handelsman, Jo, Restrepo, Silvia, Junca, Howard
• Format: Article
• Language: English
• Subjects: Functional metagenomics; GFP expression; T7 promoter; Transposon mutagenesis
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2020.01.007

•TnC_T7 transposon was constructed to improve the efficiency of metagenomic functional screenings.•TnC_T7 promotes inducible transcription of foreign DNA by expressing the T7RNA polymerase.•Random T7 promoter insertion by TnC_T7 was validated in both plasmid and fosmid DNA.•TnC_T7 favored gene expression by inserting up to 8.7 kb upstream from the gene of interest. 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 (PBAD), 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.