Cloning strategy for recovering phage-displayed Cry1Aa13 mutants from phages with affinity towards proteins present in the gut of Ceratitis capitata

Abstract: Using the phage display technique, a pool of phages from a library of bacteriophages expressing Cry1Aa13 toxins with modified loops 2 at the domain II was selected that showed affinity toward proteins present in the guts of the Mediterranean fruit fly, Ceratitis capitata. The sequences of the hypervariable regions of the in vivo selected phages were analysed and an almost identical sequence was obtained in all of the selected phages. Those phages bearing toxins different from the wild type toxin at the loop 2 were selected in order to recover the Cry1Aa13 mutant toxins. Here we describe the cloning strategy designed and used to clone the toxins from the phage genome in order to be expressed.