Primers used in this study are listed in Table 2 . All fragments were amplified with oligomers having uracil incorporated, using the Phusion U polymerase (Thermo Scientific). The plasmids and promoter library were constructed by the uracil-specific excision reagent (USER) cloning method (Geu-Flores et al. 2007 (link); Nour-Eldin et al. 2006 (link)). In brief, 1 μl of 5× HF buffer (Thermo Scientific) and 1 U of USER™ enzyme mix (New England Biolabs, 1 U/ml) were added to 10 µl of the mixture of purified PCR products, plasmid backbone, or genes.
The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. 8 µl of water was added to the reactions, reaching a final volume of 20 µl. 5 µl diluted USER mixture was used to transform chemically competent E. coli TOP10 cells (Thermo Scientific) (Sambrook and Russell 2001 ).
The reaction mixture was incubated for 25 min at 37 °C, followed by 25 min of incubation at a temperature optimized for annealing of the fragments for 25 min. 8 µl of water was added to the reactions, reaching a final volume of 20 µl. 5 µl diluted USER mixture was used to transform chemically competent E. coli TOP10 cells (Thermo Scientific) (Sambrook and Russell 2001 ).
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