Yeast display vector, Clones containing premature stop codons cannot survive.

Yeast display vector, clones containing premature stop codons cannot survive.. The Leu2 gene is deleted in EBY100 cells. Therefore the cells cannot survive unless leucine is available from either plasmid or culture medium. If reading frame shifts or premature stop codons (PSC) exist in the expression cassette, no Leu2 expresses, and the EBY100 cells won't survive in the leucine-deficient medium. When antibody libraries are synthesized with mixed bases, the majority of clones contain PSC, which cannot normally display protein. The PSC-containing clones will keep growing in the leucine-sufficient medium but not in the leucine-deficient medium (Figure 2). One scFv gene was cloned into pYSD-C/N and pYSD-C/N-Leu2 vectors. First, they grew in a tryptophan-deficient medium (Teknova # C8130), then they were induced in C9115 and C9220. pYSD-C/N transformants cannot grow in C9220. pYSD-C/N-Leu2 transformants grew well in the C9220 medium, and the display and binding affinity were not affected (Figure 2).