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. Author manuscript; available in PMC: 2009 May 2.
Published in final edited form as: J Mol Biol. 2008 Mar 4;378(3):653–665. doi: 10.1016/j.jmb.2008.02.058

Figure 5. Assessment of engineered monomer heterospecificity using a qualitative assay.

Figure 5

(a)–(c) Varying concentrations of 2:1 Cre-Lox complex, indicated above each lane in nanomolar, were reacted with ~ 15 nM supercoiled Lox-containing pLITMUS plasmid for 16 hours44 (see MATERIALS AND METHODS), electrophoresed through 1.2 % agarose gels and stained with ethidium bromide. The major products a complex mixture linear monomers and multimers (white arrows, upper left panel), as well as catenanes and plasmid topoisomers. Single site restriction enzyme digests yield the expected pair of product bands and a single reactant band (not shown).

(a) CreAA/LoxP x LoxP (middle) and ALSHG-F/LoxM7 x LoxM7 (right) compared to CreWT/LoxP x LoxP (left) reactions. The lack of complete conversion of supercoiled substrate suggests a greatly reduced recombination and//or topoisomerase activity. ALSHG-F was completely inactive in these assays. CreAA (left) or ALSHG-F (middle) alone exhibited no recombination activity in chimeric substrate (b) LoxM7P x LoxM7P or (c) LoxPM7 x LoxPM7 reactions, but the 1:1 CreAA+ALSHG-F mixture (right) recombined both substrates. However, LoxPM7 x LoxPM7 reactions were much less efficient.