UW-Madison researchers have developed three novel antibiotic-free plasmid positive selection mechanisms.
1. Phage repressor protein R: The first system is a derivative of plasmid pSIP411 encoding the phage repressor protein R (pSIP-eftu-R). The inventors’ model strain is Limosilactobacillus reuteri ATCC PTA 6475 (L. reuteri), which contains two prophage genomes, as do many bacteria. The repressor protein represses induction of prophages from the L. reuteri chromosome, and the inventors found that expression of the phage repressor provides a strong selective pressure, which resulted plasmid maintenance for at least 200 generations in antibiotic-free media.
2. SucP: The inventors constructed L. reuteriΔsucP by deleting the gene sucP, which encodes the enzyme sucrose phosphorylase, which hydrolyzes sucrose. L. reuteriΔsucP cannot grow in media with sucrose as a sole carbon source (SM). The inventors plan to clone the sucP gene into a plasmid to enable the mutant strain will retain the plasmid in sucrose-containing media.
3. AdhE: Analogously, when the inventors inactivated the gene encoding the bifunctional alcohol/aldehyde dehydrogenase (AdhE) enzyme in L. reuteri, the resulting strain L. reuteri ΔadhE could not grow in media with glucose as a sole carbon source (GM). However, by inserting the adhE gene into the plasmid, the inventors can restore the function of adhE and ensure stable maintenance of the plasmid in GM.
These antibiotic-free plasmid selection systems provide alternative methods for selecting and maintaining plasmids without relying on antibiotic resistance genes.