Construction and Analysis of a Promoter-trap Library of Streptococcus pneumoniae Which Employed gfp as a Reporter Gene

Abstract:

Firstly a suicide plasmid pEVP3-SDGFP which employed gfp as a reporter gene was constructed. DNA fragments of S. pneumoniae TIGR4 were cloned upstream of the promoterless green fluorescence protein (gfp) gene in pEVP3-SDGFP and a plasmid library which includes 58000 recombinants was constructed. Considering insert DNA orientation and insert size, this library represents 5 coverages of the 2.2 Mb S. pneumoniae genome. 90% of these clones had DNA fragments of S. pneumoniae and the library is random. Then this plasmid library was transformed into TIGR4. Through recombination, the plasmid DNA which includes the random DNA fragments was placed behind the homologous sequence of the genomic DNA of S. pneumoniae. The recombinants were screened according to the antibiotic gene in plasmid, and the S. pneumoniae library was obtained. This library includes 500000 S. pneumoniae transformants. Analysed by fluorescence microscope and flow cytometry, this S. pneumoniae library contains both the in vivo-induced gene fragments and in vitro-expressing fragments which can be reported by GFP. So this promoter-trap library can be used in analyzing the in vivo-induced gene of S. pneumoniae by differential fluorescence induction.