Figure 4

Complementation experiments of triple ilv deletion mutants. Isoy16 was transformed with plasmids expressing genes for wild-type and truncated Ilv2ΔN54, Ilv5Δ48 and Ilv3Δ19 enzymes, respective combinations, and codon-optimized genes for truncated Ilv2ΔN54, Ilv5Δ48 and Ilv3Δ19 proteins. Transformants were grown on selective SMD + leu-val + ile medium, washed with sterile water and spotted in serial dilutions on selective SMD medium agar plates containing or lacking valine or isoleucine, respectively, and incubated for up to 7 days at 30°C (detailed description in Methods). Cells containing empty vectors were pregrown on selective SMD + leu + val + ile medium. Panel A: Isoy16 containing empty vectors or plasmids encoding for wild-type or truncated Ilv2, Ilv5 and Ilv3, respective combinations. Panel B: Isoy16 expressing plasmid p425-synthILV235. Isoy16 transformed with empty vector were spotted as a negative control.