Expanding the SiMPl Plasmid Toolbox for Use with Spectinomycin/Streptomycin

[Image: see text] We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPl(x)_N and pSiMPl(x)_C, which can be stably maintained in Escherichia coli with a single antibiotic x. The method exploits the split intein gp41–1 to reconstitute the enzyme conferring resistance toward the antibiotic x, whereby each enzyme fragment is expressed from one of the plasmids in the pair. pSiMPl plasmids are currently available for use with ampicillin, kanamycin, chloramphenicol, hygromycin, and puromycin. Here, we introduce another pair for use with spectinomycin/streptomycin, broadening the application spectrum of the SiMPl toolbox. To find functional splice sites in aminoglycoside adenylyltransferase, we apply a streamlined strategy looking exclusively at the flexibility of native cysteine and serine residues, which we first validated splitting the enzymes conferring resistance toward ampicillin, kanamycin, chloramphenicol, and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance toward antibiotics.