Figure 3

Experiment diagram of interaction between essential elements in this two-hybrid system. In order to examine whether interactions happen among several essential elements involved in this two-hybrid system, such as phaP promoter, DBD, PhaP, lacZ reporter gene, interaction protein partners and PHB granules. E. coli XL1-Blue harboring plasmids were cultured to detect the possible interaction between these elements by detecting β-galactosidase activity. DBD regulates LacZ expression by turning DBD on/off the DBD binding site of phaP promoter. (A) pOZ, the promoter of phaP can give rise to strong expression of LacZ; (B) pDBD-Z, DBD can suppress LacZ expression by binding on DBD binding site of phaP promoter; (C) pDBD-Z and pBHR68, PHB granules can not pull down DBD from DBD binding site, indicating DBD has no interaction with PHB granules; (D) pDBD-Z and pP-CAB, similar with (C), PhaP can not pull down DBD, indicating DBD has no interaction with PhaP; (E) pDBD-Z and pFos-P-CAB, similarly, DBD has no interaction with PhaP-bFos fusion; (F) pDBD-Jun-Z and pP-CAB, similarly, DBD-bJun has no interacton with PhaP; (G) pDBD-Jun-Z and pFos, when PHB granules are absent, the interaction of DBD-bJun and bFos can not give rise to LacZ expression; (H) pDBD-Jun-Z and pFos-P, when PHB granules are absent, the interaction of DBD-bJun and PhaP-bFos can not give rise to LacZ expression; These assays were used to eliminate the influences of unspecific interaction, which was so called background noise. X: bait protein; Y: prey protein.