| 초록 |
We developed a CRISPRi-based double inverter gate for selectively identifying bacterial cells harboring the multiple plasmids. To this end, we first generated a NOT gate through the CRISPRi plasmid (pSLiP plasmid) which inhibits cell growth by interfering with the transcription of the essential gene involved in the host growth. Next, a double inverter system, which inhibits the growth-inhibitory function of the pSLiP plasmid by another sgRNA expressed from a separate plasmid, was created to restore the host cell growth. Using this system, we enriched cells with two plasmids expressing green or red fluorescent protein, respectively, without using antibiotics. In addition, we also increased plasmid stability by the CRISPRi-based double inverter system, which resulted in enhanced production of terpenoids. Thus, the developed system paves the way in synthetic biology for applications of metabolic engineering and therapeutics production. |
