Espah Borujeni

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The genetic implementation of the UBER system combines expression of T7 RNA polymerase (T7 RNAP) and tetracycline repressor (TetR) within a self-regulating genetic circuit that uses a cross-species priming promoter, wild-type T7 promoters, an engineered T7 promoter flanked by TetO operator sites (T7-TetO promoter), cross-species translation signals and efficient transcriptional terminators (Fig. 1a).

Restriction endonucleases and DNA-modifying enzymes were purchased from New England Biolabs and used according to manufacturer’s instructions. DNA plasmid isolation was performed using GeneJET Plasmid Miniprep Kit (Thermo scientific). DNA extraction from agarose gel was carried out using the GeneJET Gel Extraction Kit (Thermo scientific). DNA sequencing was carried out by Beckman Coulter Genomics (Takeley, United Kingdom).