Figure 1
Elements used in construction of the polypeptide secretion library of S. aureus in E. coli. A. Expression vector pSRP18/0 contains an expression cassette comprised of a 5' untranslated sequence upstream of the flagellin gene of E. coli MG1655 (fliCMG1655) here indicated fliC5'UTR, a DNA fragment encoding the N-terminal 20 amino acids fliCMG1655 (fliC1-60), a synthetic FLAG tag encoding sequence (flag) and a 3' untranslated region downstream of fliCMG1655 (fliC3'UTR). EcoRV indicates the unique cloning site for foreign DNA fragments, horizontal arrows indicate the oligonucleotides used as primers for PCR (017F, 025F and 028R) and sequencing (017F and 071R) of the cloned inserts and black lines indicate sequences of the plasmid pBR322. SalI and BamHI indicate the restriction sites created during cloning of the expression cassette into pBR322. B. Agarose gel electrophoretic analysis of the chromosomal DNA isolated from S. aureus NCTC 8325-4 and used in generation of the library. The purified DNA is shown in the left lane, randomly fragmented and blunted DNA in the right lane. Ns indicate not sonicated and s stands for sonicated and polished DNA. The positions of molecular weight markers in base pairs are shown to the right.