Genetic engineering of Bt Cry1Ac toxin

PCR was performed using PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies), VeraSeq ULtra DNA polymerase (Enzymatics), or Phusion U Hot Start DNA Polymerase (Life Technologies). Water was purified using a MilliQ water purification system (Millipore). Plasmids and selection phages were constructed using USER cloning (New England Biolabs). Genes were either synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies) or amplified by PCR from native sources. Cry1ac was amplified by PCR from the B. thuringiensis strain Bt_B107284 and cloned into the Bt expression vector pMON101647 using Hot Fusion^{32 (link)} to generate the expression plasmid pMON133051, which served as a template for amplifying Cry1ac fragments for constructing PACE vectors. The toxin-binding region from T. ni cadherin (A1133-T1582, AEA29692.10), referred to as TnCAD-FL, was synthesized using 45–60-mer oligonucleotides (Integrated DNA Technologies) by overlap extension PCR using KOD Hot Start DNA polymerase (EMD Millipore). The synthetic wild-type TnCAD-FL template was used to generate the TnTBR3-FL fragment via site-directed mutagenesis using the QuikChange II kit according to the manufacturers’ instructions. (Agilent Technologies). DNA vector amplification was carried out using NEB Turbo or DH5α cells (New England Biolabs).