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Expanding the SiMPl Plasmid Toolbox for Use with Spectinomycin/Streptomycin
Navaneethan Palanisamy1,2, Jara Ballestin Ballestin1,2, Barbara Di Ventura1,2
1Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Schänzlestraße 18, 79104 Freiburg, Germany.
ACS Omega
|June 14, 2021
Summary
The SiMPl plasmid toolbox now includes spectinomycin/streptomycin resistance, expanding its utility. This system uses split inteins to reconstitute antibiotic resistance enzymes in E. coli.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Microbiology
Background:
- The SiMPl plasmid toolbox enables stable maintenance of paired plasmids in Escherichia coli using a single antibiotic.
- This system relies on the split intein gp41-1 to reconstitute antibiotic resistance enzymes, with each fragment expressed from a separate plasmid.
- Existing pSiMPl plasmids support ampicillin, kanamycin, chloramphenicol, hygromycin, and puromycin resistance.
Purpose of the Study:
- To expand the SiMPl toolbox by introducing a new plasmid pair for spectinomycin/streptomycin resistance.
- To develop and validate a streamlined strategy for identifying functional splice sites in antibiotic resistance enzymes, focusing on cysteine and serine residues.
Main Methods:
- The split intein gp41-1 was utilized to reconstitute antibiotic resistance enzymes.
- A novel strategy was employed to identify functional splice sites by analyzing the flexibility of native cysteine and serine residues within aminoglycoside adenylyltransferase.
- The strategy was initially validated by splitting enzymes conferring resistance to ampicillin, kanamycin, chloramphenicol, and hygromycin.
Main Results:
- A new pSiMPl plasmid pair conferring spectinomycin/streptomycin resistance was successfully developed and integrated into the toolbox.
- The streamlined strategy for identifying splice sites based on cysteine and serine flexibility proved effective in splitting various antibiotic resistance enzymes.
- The study demonstrates the successful application of split intein technology for antibiotic resistance gene manipulation in E. coli.
Conclusions:
- The SiMPl toolbox has been significantly enhanced with the addition of spectinomycin/streptomycin resistance, broadening its applicability in molecular biology and synthetic biology.
- The validated strategy for splice site identification offers a powerful approach for engineering split enzymes and could be applied to other antibiotic resistance genes.
- This work facilitates the development of novel genetic tools for research in microbiology and biotechnology.
