Team:TCU Taiwan/SystemDesign

We synthesised a new CIRSPR system based on a CRISPR designed by Standford-Brown 2013 (BBa_K1218011). Here we analyzed its sequence and then removed its Cas9 protein with Cas9’s promoter because we want our Cas9’s expression can be inducible. So then we synthesized a new  Cas9 structure which contains an inducible promoter, pRha (BBa_K914003). This promoter can be induced by rhamnose and silenced by glucose, both sugar can be find in nature. Next, we put our inducible Cas9 back to the CRISPR system, and because tracrRNA and crRNA have no function without Cas9 protein, this CRISPR is now under our control.

After constructed our CRISPR system, we ligated it with a red fluorescent reporter gene (BBa_I13521), so once our system is spread, all bacterium get this system will become red. Finally, we put whole system into phagemid pBluescript II SK(-), and get the recombinant phagemid for M13 infection.