PT  - JOURNAL ARTICLE
AU  - Palanisamy, Navaneethan
AU  - Ballestin Ballestin, Jara
AU  - Di Ventura, Barbara
TI  - Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin
AID  - 10.1101/2021.02.04.429595
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.04.429595
4099  - http://biorxiv.org/content/early/2021/02/04/2021.02.04.429595.short
4100  - http://biorxiv.org/content/early/2021/02/04/2021.02.04.429595.full
AB  - We recently developed the SiMPl plasmid toolbox, which is constituted by pairs of plasmids, generically indicated as pSiMPlx_N and pSiMPlx_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 towards 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 towards ampicillin, kanamycin, chloramphenicol and hygromycin. This strategy could be used in the future to split other enzymes conferring resistance towards antibiotics.Competing Interest StatementThe authors have declared no competing interest.