Figure 1.
Schematic representation of the expression cassette and the library designs. (A) Block diagram of the expression cassette showing (i) the E. coli lac promoter (Plac), (ii) the E. coli trp operon-derived SD sequence (SDtrp), (iii) the gene sequence corresponding to either the eight first amino acids of the wild-type TrpL peptide (reference vector), or the corresponding variegated sequence window of any library member from ExLib1 or ExLib2, (iv) the gene encoding the IgG-binding Z domain (Z) and (v) the gene encoding the EGFP. The two alternative start codons are indicated (Start 1 and Start 2); (B) DNA and deduced amino acid sequence of the translation initiation region of the reference vector pBR-TrpL-ZEGFP discussed in the text. Recognition sites for restriction enzymes discussed in the materials and methods section are indicated as underlined; (C) Description of the randomization design used to construct the ExLib1 library, showing location and nature of nucleotide variations introduced via the use of the two degenerate oligonucleotides Lib-1a and Lib-1b. Note: In order to be able to include all six codons for Leu at position +7, two different oligonucleotides (Lib-1a and Lib-1b) were used in the library construction (in a 2:1 mixture); (D, E) Description of the randomization design used to construct the ExLib2 library, showing location and nature of nucleotide variations introduced via the use of the degenerate oligonucleotide Lib-2 (N = A, G, C or T). The design of this library allowed for two in vivo scenarios, involving either a translational start at the first start codon (underlined in Figure 1D) or at the second start codon (underlined in Figure 1E). See text for details.