Screening of promoters from rhizosphere metagenomic DNA using a promoter‐trap vector and flow cytometric cell sorting

Abstract

We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers.

Gentisate 1,2-dioxygenase, in the third naphthalene catabolic gene cluster of Polaromonas naphthalenivorans CJ2, has a role in naphthalene degradation

“…Plasmid pK19mobsacB (Schäfer et al, 1994), containing oriT for conjugative mobilization and sacB (encoding the Bacillus subtilis levansucrase) as a counter-selectable marker, was used to construct three individual nagI knockout mutants in strain CJ2. Mutagenic plasmids pK19mobsacBDnagI1, pK19mobsacBDnagI2 and pK19mobsacBDnagI3 with 2 bp deletions in the nagI genes were constructed via a three-step round-PCR approach (Lee et al, 2011) as shown in Fig. 1a.…”

“…Plasmid pK19 mobsacB (Schäfer et al , 1994), containing oriT for conjugative mobilization and sacB (encoding the Bacillus subtilis levansucrase) as a counter-selectable marker, was used to construct three individual nagI knockout mutants in strain CJ2. Mutagenic plasmids pK19 mobsacB Δ nagI1 , pK19 mobsacB Δ nagI2 and pK19 mobsacB Δ nagI3 with 2 bp deletions in the nagI genes were constructed via a three-step round-PCR approach (Lee et al , 2011) as shown in . Briefly, nagI gene PCR amplicons with a 2 bp deletion and Hin dIII and Eco RI sites at the flanking regions were generated by PCR using two primer sets each, nagIx1F ( Hin dIII)/nagIx1R and nagIx2F/nagIx2R ( Eco RI) shown in , where ‘x’ indicates 1, 2 or 3 for nagI1 , nagI2 and nagI3 , respectively, and then were digested with Hin dIII and Eco RI (New England Biolabs).…”

Advancing Weed Management Strategies Using Metagenomic Techniques

“…Another example of indirect activity assay is through promoter-trap vectors where the vector contains the GFP gene but no promoter. Therefore, GFP will not be expressed until a promoter is present in the DNA fragment (Dunn et al 2003;Lee et al 2011).…”

“…For example, promoter-trap vectors have been applied to search for promoters responding to naringenin, a compound released by plant roots. FACS was used following additions of naringenin to find genes induced by the compound (Lee et al 2011). As cell sorting technology improves, phenotypes other than fluorescence could be added to the molecular toolbox.…”

Metagenomic Analysis of Contaminated Soils

“…A detailed protocol for SIGEX has been published (Uchiyama & Watanabe, 2008). However, to date, few follow up studies -except for those from the original authors (Uchiyama & Miyazaki, 2010;Uchiyama & Watanabe, 2007) -have used SIGEX for metagenomic analyses (Lee et al, 2011). Nonetheless, it is recurrently cited as a method with excellent potential for recovering novel operons from metagenomic clone libraries (Daniel, 2005;Ekkers et al, 2012;Simon & Daniel, 2011;Taupp et al, 2011;Wexler & Johnston, 2010;Yun & Ryu, 2005).…”