RT Journal Article
SR Electronic
T1 Expanding the SiMPl plasmid toolbox for use with spectinomycin/streptomycin
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.02.04.429595
DO 10.1101/2021.02.04.429595
A1 Palanisamy, Navaneethan
A1 Ballestin Ballestin, Jara
A1 Di Ventura, Barbara
YR 2021
UL http://biorxiv.org/content/early/2021/02/04/2021.02.04.429595.abstract
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.