Fig. 3.

SMPL mutagenesis with test sequences. Mutated codons are in bold, insert codons are italicized and adenine containing codons of the tr are underlined. (A) To determine the viability of an insert C-terminal to Mtd position 367, a heterologous, non-adenine insert encoding a restriction site was constructed. A silent mutation (GCG→GCT) was also inserted at position 367 as a non-adenine marker for transfer. The tropism switching mechanism repaired the silent mutation and excised the restriction site. (B) To test a larger insert in position 367, a 14 codon sequence was inserted into position 367 and the entire insert was transferred to the VR. Both heterologous and endogenous adenine containing amino acids were mutated before insertion into VR, as shown. (C) The insertion of a His₆ peptide at position 349 tested this region of the Mtd for both variability and viability. Seventeen percent of endogenous adenine containing amino acids were mutated; however, no heterologous mutagenesis occurred. (D) To test the ability of the DGR to mutate heterologous adenines in position 348, a 25 amino acid sequence was inserted into the tr. Nineteen codons of the insert were transferred in-frame to the vr with mutagenesis throughout the insert and surrounding scaffold.