Construction of promoter-trap library screening in vivo-induced gene in Streptococcus pneumoniae / 生物医学工程学杂志

ABSTRACT

To identify in vivo-induced gene in Streptococcus pneumoniae (S. pn) through a novel in vivo expression technology (IVET), a large promoter-trap library using galU and lacZ as the reporters was constructed. Based on the suicide vector pEVP3, a new vector pEVP3-galU was constructed with promoterless galU gene as an in vivo reporter. Firstly, promoterless galU gene was directly cloned into pEVP3 fusing with promoterless lacZ gene (an in vitro reporter). Then the random pieces of S. pn chromosomal DNA (200-500 bp), obtained by partial Sau3AI restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Upon introduction of the ligated plasmid library into E. coli DH5alpha by transformation, about 70,000 recombinants were recovered. Considering insert DNA orientation and insert size, this represents 5 coverage of the 2.2 Mb S. pn genome; 90% of these clones had 250- to 500-bp inserts. Thus, the library retained maximal complexity. Transformation by this plasmid library yield 450,000 S. pn transformants. The library was used to infect animals in intraperitoneal model. Those strains survived in vivo while exhibiting a white colony phenotype on TSA agar containing X-gal would indicate that the DNA fragment upstream of the galU reporter contained an in vivo-induced promoter. The promoter-trap library is suitable for screening in vivo-induced gene of S. pn.