pI-SceI was constructed by cloning the I-sceI nuclease from pSLTS [a gift from Shelley Copley, Addgene plasmid #59386, (61 (link))] cloned into the expression vector pHERD30T between the EcoRI and XbaI sites using primers I-SceI F and I-SceI R (Table 2). Restriction cloning sites in primer sequences are indicated by capital letters. pCut was constructed by cloning the I-SceI recognition sequence (TAGGGATAACAGGGTAAT) into pUCP18 between the HindIII and EcoRI sites. pEmpty is simply the empty pUCP18 vector. Primers used to screen for the presence/absence of circular and linear plasmid DNA are as follows: pCutF and M13R produce a 1-kb product indicating the presence of circular pCut (these primers will not amplify linearized pCut DNA). pCutF and pCutR produce a 0.5-kb product indicating the presence of both linear and circular forms of pCut. Clones carrying pI-SceI and confirmed linearized pCut plasmid DNA or those carrying pEmpty were grown with or without 50 mM putrescine in LB broth for the indicated times. Intracellular polyamines were measured as described above.