DNA vectors
A new series of DNA vectors designated pDuEx (plasmid Dual Expressions) were constructed. The vectors used in our assay, pDuEx-Bait (B or B2) and pDuEx-Prey (P) have five characteristics. And the dual gene expression vector. pDuEx-Bait-Prey vector can express similar levels of bait- and prey-genes.
pDuEx-Bait (pDuExB;GeneBank# EF565884, pDuExB2;GeneBank#EF565885)
pDuEx-Prey (pDuExP;GeneBank# EF565883)
- pDuEx carries the Gateway recombinant site (No.1) for cDNA subcloning.
- pDuEx carries Cre-loxP recombinant sites (No.2) for dual gene expression.
- pDuEx carries split Renilla luciferase (No.3).
- Split Renilla luciferase contains hexamer peptide tags (Tetra-Cys tag;No.4, 6xHis;No.5).
Dual gene expression vector
Gene expression levels of bait and prey genes in the dual gene expression vector
| pDuEx Cloning procedure | |
| Day 1 | LR reaction |
| Day 2 | Picking up colonies from agar plates and broth-culture |
| Day 3 | Plasmid Mini Prep |
| Day 4 | Picking up colonies from agar plates and broth-culture |
| Day 5 | Plasmid Mini Prep |
It took 5 days to obtain final pDuEx-Bait-Prey vectors after obtaining pENTR clones. In the cloning procedure, we randomly picked 2-3 colonies after bacterial transformations with Gateway reaction mixture (Day 2). By confirming the proper recombination with restriction enzyme digestion (PvuII) followed by the gel electrophoresis, single clones from pDuEx-Bait and pDuEx-Prey were used to conduct the Cre-loxP reaction. Two to three bacterial colonies were randomly selected from each plate (Day 4) and the proper recombination was confirmed with a restriction enzyme digestion (EcoRV or PvuII) followed by the gel electrophoresis. A bacteria colony carrying a proper pDuEx-Bait-Prey vector was then cultured over night in a 100 ml culture medium to obtain about 100 µg of the purified vector.