Amplimer planning for PIPE cloning. Gene Composer was used to plan the virtual and PIPE cloning of full-length and terminal deletion constructs of a codon engineered Bacillus subtilis FtsZ gene. A, PIPE cloning is illustrated wherein the synthetic gene insert (orange) is amplified by designed forward (red-orange lines) and reverse (orange-blue lines) primers to generate insert PCR material (iPCR, ~900–1350 bp). The expression vector is amplified with reverse (red-black lines) and forward (blue-black lines) primers to generate vector PCR material (vPCR, full length is ~5.8 Kbp). The terminal sequences iPCR products are complementary to the terminal sequences of vPCR products (red of iPCR complements red of vPCR and blue of iPCR complements blue of vPCR). This allows the iPCR and vPCR products to anneal to form circular DNA molecules that are replicated upon transfection into host bacteria. B, Based on alignment analysis of the target amino acid sequence for FtsZ to relevant PDB entries, terminal truncation points were selected (denoted by vertical bars in the base construct view). Permutations of the terminal deletions were used to generate multiple constructs that were virtually cloned according to PIPE parameters in Gene Composer (listed in the vector construct tab, below the base construct view). C, Agarose gel analysis of PIPE Cloning constituents. After the terminal deletions of FtsZ-encoding constructs were designed and virtually cloned into the expression vector. Gene Composer generated a spreadsheet file of all necessary amplification primers to produce iPCR and vPCR products. This oligonucleotide file was uploaded directly to the oligonucleotides synthesis vendor website together with information dictating the specific positions of the designed oligos in a 96-well plate. The, lyophilized oligos were resuspended and used directly in an automated PIPE (polymerase incomplete primer extension) cloning procedure on a Tecan EvoFreedom liquid handling robot. Agarose gel analysis of the iPCR in lanes 1–6, and vPCR in lane 7 products are shown together with molecular size standards.